Dissertations / Theses on the topic 'Metabolomics'

As life expectancy has risen steadily over the last years and diseasefree aging is more and more challenging, understanding the complexity of age and aging is of great importance. Metabolomics is one of the novel approaches in systems biology with high potential to deliver answers to these questions. However, only a few metabolic studies based on large samples are available so far. In this thesis, I present results from two population-based studies, the German KORA Follow-Up 4 (KORA F4) study as a discovery cohort with a sample of 1,038 female and 1,124 male healthy participants (32–81 years) and 724 healthy females from UK Adult Twin Registry (TwinsUK) as replication. Targeted metabolomics of fasting serum samples with flow injection analysis coupled with tandem mass spectrometry (FIA-MS/MS) positively quantified 131 metabolites after stringent quality control. Among these, 71 and 34 metabolites were significantly associated with age in females and males, respectively, after adjustment for body mass index (BMI), which is highly correlated (r=0.9) with age. These results indicate that metabolic profiles are age dependent and sex specific. Then, a set of the 12 most age-discriminative, independent metabolites was identified in women with an approach based on random forest and stepwise variable selection. This set showed highly significant differences between subjects aged 32–51 years and 52–77 (p-values range 1.3E-09 – 1.9E-46, significance threshold p=0.004). Ten out of these 12 metabolites replicated in unrelated females from the TwinsUK study, including five metabolites the concentrations of which increased with age (C12:1, C18:1, sphingomyelin (SM) C16:1, SM C18:1 and phosphatidylcholine (PC) aa C28:1), while histidine decreased gradually. Three glycerophospholipids (PC ae C42:4, PC ae C42:5, PC ae C44:4) showed declines around the age of 51 years. Meta-analysis of both studies gave virtually the same results as KORA alone. These observations might reflect many different processes of aging such as incomplete mitochondrial fatty acid oxidation, counteracting oxidative stress, and changes in vascular functions. The identification of these ten age-related metabolites should help better understand aging pathways and networks and with —more discoveries in the future— eventually help enhance healthy aging and longevity.

Metabolomics is a rising field combining bioinformatics and cheminformatics together. A major component of research is having a reliable data source, which usually comes in the form of metabolomic databases. This paper documents arising issues revolving categorizing metabolome compounds within databases, and a possible solution in the form of an R package that is capable of matching up various metabolome identifiers that originate from various metabolome databases. Then, by using this package we reflect on the average coverage of external reference between metabolome databases to highlight the lack of a universal compound primary identifier.

The thesis presentation was held over Zoom due to the recent COVID19 pandemic.

Background Rheumatoid arthritis (RA) is associated with both local and systemic inflammation which influences the function of the whole body as well as local tissues in the joints. Significant consequences of this are changes in metabolism. Hence, we hypothesised that chronic inflammation alters metabolism and that the metabolic profile of an individual patient with early inflammatory arthritis predicts the subsequent course of disease. Furthermore, we suggested that these metabolic changes would identify biomarkers of response to treatment in inflammatory arthritis and provide novel insights into disease mechanisms. Methods Using NMR spectroscopy of serum, urine and synovial fibroblasts we derived metabolic profiles and subjected these to multi-parameter analyses to identify metabolic differences associated with inflammation. Results We were able to predict outcome in patients with early arthritis using material derived from cultured synovial fibroblasts but were unable to do so using serum. There was a significant association between CRP levels in the patients’ serum and the metabolic profile of their synovial fibroblasts and their serum. There was also a significant association between the metabolomic fingerprint of synovial fibroblasts and the fibroblasts’ IL6 production. We found differences in metabolites between urine samples of RA and psoriatic arthritis (PsA) patients and were able to predict responses to anti-TNF therapy in patients with RA. Discussion Our results demonstrate that underlying inflammatory processes drive significant changes in metabolism that can be measured in the peripheral blood, synovial fibroblasts and urine samples in patients with inflammatory arthritis.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008.
Includes bibliographical references (p. 153-160).
A primary challenge in embryology is to understand the factors that govern the development of preimplantation (PI) embryos and how these factors relate to embryo viability in the field of in vitro fertilization (IVF). This is particularly important as clinical policy moving towards single embryo transfer (SET) has gained awareness to manage unprecedented numbers of multiple births, such as twins and triplets, resulting from artificial reproductive techniques. Conditions that correlate with developmental potential of candidate embryos are disputed in the field, however, as the requisite data is difficult to obtain.The metabolic profiles of embryos during in vitro culture have been suggested as a key indicator of developmental potential, and approaches have been clinically implemented to select transfer candidates which make the most efficient use of nutrients. Existing microdroplet analysis techniques are accurate and suitable for non-invasive assessment of single embryos. Unfortunately, the process of determining metabolite levels in nanoliters of culture media through fluorometric assays is low-throughput and requires specialized expertise, hindering widespread clinical use of these methods. The goal of this thesis is to develop microfluidics-based approaches for improving metabolic analysis of PI embryos and mammalian cells. This challenge necessitates two competencies: methods for automating chemical assays and methods for supporting cell cultures, which can be integrated with analysis. Contributions include a standalone platform for determining the metabolite use of single embryos. Profiles may be acquired automatically, which reduces significant technician hours and improves repeatability. Techniques are developed for performing embryo culture in the smallest culture volumes to date in microfabricated environments. Microfluidic approaches have enabled culture that outperforms the current state of art approach based on cell count measurements.
(cont.) An integrated system is introduced, merging analysis and culture competencies to perform metabolic profiling of separate cultures of mammalian cells in parallel. Finally, new paradigms in microfluidic design are presented based on the concept of vertically integrated architectures, suitable for overcoming density limitations of microfluidic assays. A scalable analysis platform for refining embryo selection has been long warranted and would enable pursuit of the difficult questions relating metabolism and embryo viability as the clinical movement towards SET continues.
by John Paul Urbanski.
Ph.D.

Metabolomics lies at the fulcrum of the system biology 'omics'. Metabolic profiling offers researchers new insight into genetic and environmental interactions, responses to pathophysi- ological stimuli and novel biomarker discovery. Metabolomics lacks the simplicity of a single data capturing technique; instead, increasingly sophisticated multivariate statistical techniques are required to tease out useful metabolic features from various complex datasets. In this work, two major metabolomics methods are examined: Nuclear Magnetic Resonance (NMR) Spec- troscopy and Liquid Chromatography-Mass Spectrometry (LC-MS). MetAssimulo, an 1H-NMR metabolic-profile simulator, was developed in part by this author and is described in the Chap- ter 2. Peak positional variation is a phenomenon occurring in NMR spectra that complicates metabolomic analysis so Chapter 3 focuses on modelling the effect of pH on peak position. Analysis of LC-MS data is somewhat more complex given its 2-D structure, so I review existing pre-processing and feature detection techniques in Chapter 4 and then attempt to tackle the issue from a Bayesian viewpoint. A Bayesian Partition Model is developed to distinguish chro- matographic peaks representing useful features from chemical and instrumental interference and noise. Another of the LC-MS pre-processing problems, data binning, is also explored as part of H-MS: a pre-processing algorithm incorporating wavelet smoothing and novel Gaussian and Exponentially Modified Gaussian peak detection. The performance of H-MS is compared alongside two existing pre-processing packages: apLC-MS and XCMS.

Metabolites are a potentially useful source of detecting and identifying disease specific biomarkers. This thesis investigates the possibility of using metabolomics applications to detect Alzheimer’s disease associated metabolite peaks in patients and to detect longitudinal changes of the disease. Serum samples and clinical data were collected from 60 healthy controls and 60 Alzheimer’s disease patients (60 at baseline and 60 at 12 month follow-up). The metabolic fingerprinting of serum samples using the FT-IR lacked discriminatory power to discriminate Alzheimer’s disease and non-disease samples due to the similar magnitude of biological and analytical variation. The metabolic profiling of serum samples using the GC-ToF-MS did not reveal any significantly altered metabolite peaks between the Alzheimer’s disease and non-disease groups. Metabolic profiling of serum samples using the UPLC-LTQ/Orbitrap-MS operated in the positive ionisation mode did not reveal any significantly altered metabolite peaks between the disease and non-disease groups. Up to twelve metabolite peaks were significantly altered in the Alzheimer’s disease baseline and follow-up samples, indicating a potential association with disease progression. Metabolic profiling of serum samples using the UPLC-LTQ/Orbitrap-MS operated in the negative ionisation mode did not reveal any significantly altered metabolite peaks between Alzheimer’s disease and non-disease groups. Three metabolite peaks were significantly altered in the Alzheimer’s disease baseline and follow-up samples, indicating a potential association with disease progression. Metabolic profiling of serum samples with the UPLC-LTQ/Orbitrap-MS may potentially be used to detect disease and disease progression associated metabolite peaks. The metabolite peaks require identification followed by a validation experiment.

Nutrition is fundamentally important for human health and development; in particular, an adequate supply of macronutrients such as carbohydrates, lipids and proteins as well as specific elements in the form of vitamins and minerals. An insufficient intake of macronutrients or micronutrients leads to poor metabolism and physiological changes associated with a variety of negative health outcomes. Maternal nutrition is perhaps the most important determinant in foetal development in all mammalian species including humans. Additionally, nutrition in pregnancy can have long term effects on the developing infant and has been associated with development of chronic disease later in life. Changes in micronutrient status have been associated with gestational complications such as gestational diabetes mellitus (GDM), preeclampsia (PE), foetal growth restriction (FGR), and preterm birth. Pregnancy disorders such as these are poorly understood despite extensive research, and novel research into this field is desperately required. Recent advancements in technology have allowed the simultaneous measurement of large numbers of elements and metabolites. Currently, we only know how a handful of elements affect pregnancy outcomes, even though many are likely to be essential to human development. This research aimed to determine the elemental nutrition status of pregnant woman and correlate this with (1) weeks gestation, (2) pregnancy health and (3) mechanisms that may contribute to disease, with the ultimate goal of using these values to determine gestational outcomes. This was to be conducted through series of cohort analysis, providing detailed baseline information on micronutrient levels in non-pregnant and pregnant Australians, to then determine if aberrant changes in nutrition are present, and how this may contribute to any disease state. It was critical to establish a means of multi-elemental analysis on ICP-MS that was reproducible and conformed to rigorous quality control standards, ensuring the validity of measurements. This was done through the use of placental cord blood plasma, and in collaboration with an externally accredited laboratory for elemental measurements, ensuring the laboratory facilities and ICP-MS methodologies in place at Griffith University are comparable to a nationally accredited analytical facility. For post method validation, elements within a healthy population had to be determined, to then discern what can be considered abnormal. Elemental reference ranges were established in healthy non-pregnant donors attending the Red Cross Blood Bank (Queensland Red Cross Blood Service). Generally, the levels of elements were comparable to other countries and within published reference ranges. Age was associated with increases in Fe, Se, Cd, Cs, and Pb; whilst Cu, I, and Tl decreased. Sex specific differences included higher levels of Mg, K, Fe, Zn, Se, Rb, Cs, and Pb in males; whilst females had higher concentrations of Co, Cu, As, and Cd. What was interesting, was the varied concentrations of some elements between some regions within the same country, a trend that would be repeated across different studies in this thesis. Having measured the concentrations of elements in a non-pregnant population, it was essential to quantify elements in pregnant women across gestation for comparison and to determine what may be considered normal for gestation. Due to the constant growth and change that is associated with pregnancy, non-pregnant, 18-, 28-, and 36-week pregnant women were tested. There were no signs of elemental deficiencies, or over nutrition in either the non-pregnant or pregnant samples in these studies; nor was there any evidence of pollutants, contaminants, or abnormal levels of heavy metals. It was established that the changes that occur across gestation are extensive, with concentrations varying throughout gestation. However, it appears that there are a number of key factors that may influence these gestational concentrations that need to be considered, namely environmental exposures and collection methods. Further this project then highlighted changes of elements that occurred across gestation, and in pregnancy outcomes such as PE, GDM, FGR, and preterm birth. It assessed the use of elements as a means of predicting gestational outcomes at 18 and 36-weeks, indicating that earlier gestation measurements may provide a better means of discrimination, though validation is required in order to confirm these results. This thesis also evaluated small molecule metabolites and their ability to characterise and differentiate, non-pregnant, healthy pregnant, and poor gestational outcomes, noting significantly better results than elemental analysis. Specifically, in discrimination capabilities with data analysis models, small molecule metabolites outperformed the models constructed based on elements. Although further validation is required before these methods could be applied clinically. Knowledge of the influence of region, collections methods, and time of sample collection were also highlighted through cohorts from across Australia. This thesis proposes the use of a standard operating procedure for elemental and metabolic analysis to ensure the consistency of measurements, aiming to minimise preventable variance in the future.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Bilharziasis, a disease that is a major health problem in tropical and subtropical countries, is caused by worms of the genus Schistosoma. The main disease–causing species are S. haematobium, S. mansoni and S. japonicum. Bilharziasis is endemic in South Africa, mostly located in the north–east and covers one quarter of the country, with S. haematobium being the most common species. In this investigation we investigated the schistosome–induced changes in human hosts. We systematically investigated the dynamic metabolic profile of humans infected with S. haematobium using an untargeted gas chromatography?mass spectrometry (GC–MS) metabolomics approach, including univariate and multivariate data analysis. The analysis of host urinary composition is a well suited approach to understand the holistic metabolic responses to infections, since metabolomics is a branch of science concerned with the metabolite composition of biological systems and its dynamic response to both endogenous (i.e. physiology and development) and exogenous (i.e. environmental factors and xenobiotics) stimuli. As a holistic approach, metabonomics detects, quantifies and catalogues metabolic processes of an integrated biological system. In this investigation we selected the organic acid component of the metabolome for the metabolic profiling. Organic acids were determined from urine samples obtained from humans infected with S. haematobium and a control group of non–infected humans. These metabolites were quantified and identified using an automated mass spectral deconvolution and identification system (AMDIS) from which complex two–dimensional data–matrix sets were created, including assessment of the repeatability in generating a metabolomics matrix. Data matrices were analyzed by principal component and partial least square discriminant analyses (PCA and PLS–DA) to investigate which perturbations existed between the two experimental groups. The biochemical interpretation of the information from these analyses indicated that the main biochemical effects of a S. haematobium infection in humans consisted of reduced energy metabolism, liver–function disturbances and perturbations in the gut microbial population common to infections caused by other schistosoma species. Alterations of metabolites of the phenylalanine–tyrosine pathway, including aspects of catecholamine metabolism seems to be novel to a S. haematobium ii infection and hasn’t been reported in current literature. Finally, proposals were formulated for future investigations on S. haematobium infection.
Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2012.

Metabolomics provides an untargeted strategy, which has been used to evaluate the effect of dietary components on human metabolism. Epidemiological evidence indicates that increased consumption of wholegrain foods is positively associated with decreased prevalence of chronic diseases. The aim of the current work was to evaluate the application of NMR metabolomics in nutrition research, using urine and plasma samples from two short-term (postprandial) studies that evaluated responses in men and women to minimally processed wheat bran and aleurone meals, and aleurone processed into bread. The first investigations assessed the application of NMR metabolomics to assess urinary responses while the later investigations assess plasma responses in men and women consuming single meals of minimally processed wheat bran and aleurone. Results showed that there was discrimination between the urinary metabolite profiles at both one and two hours after the consumption of aleurone and bran meals compared to control meal, while there was discrimination after the consumption of aleurone meal compared to the control meal only at one hour in the plasma metabolite profiles. However, there was no clear discrimination between the aleurone and bran meals. The final investigations, which assessed the urinary responses in men and women consuming single meals of aleurone fraction incorporated into bread, showed that there was discrimination between the urinary metabolite profiles only at two hours after the control bread compared to aleurone bread. Furthermore, there were differences in urinary and plasma metabolite profiles between females and males after meal consumption throughout suggesting that gender-related differences may be important, and may need to be taken into account in metabolomics studies that assess postprandial effects. This is the first human metabolomics study exploring the gender-related differences of wholegrain meal consumption. Overall, the results indicated that metabolomics has been successful in articulating the effects of wholegrains and gender-related differences.

The growing problem of insecticide resistance is jeopardising current pest control strategies and current insecticide development pipelines are failing to provide new alternatives quickly enough. Metabolomics offers a potential solution to the bottleneck in insecticide target discovery. As a proof of concept, metabolomics data for permethrin exposed Drosophila melanogaster was analysed and interpreted. Changes in the metabolism of amino acids, glycogen, glycolysis, energy, nitrogen, NAD+, purine, pyrimidine, lipids and carnitine were observed along with markers for acidosis, ammonia stress, oxidative stress and detoxification responses. Many of the changed metabolites and pathways had never been linked to permethrin exposure before. A model for the interaction of the observed changes in metabolites was proposed. From the metabolic pathways with the largest changes, candidate genes from tryptophan catabolism were selected to determine if the perturbed pathways had an effect on survival when exposed to permethrin. Using QPCR it was found that all genes in the entire pathway were downregulated by permethrin exposure with the exception of vermilion suggesting an active response to try and limit flux through tryptophan catabolism during permethrin exposure. Knockdown of the tryptophan catabolising genes vermilion, cinnabar and CG6950 in Drosophila using whole fly RNAi resulted in changes in susceptibility to permethrin for both topical and oral routes of exposure. Knockdown of the candidate genes also caused changes in susceptibility when the insecticides fenvalerate, DDT, chlorpyriphos and hydramethylnon were orally administered. These results show that tryptophan catabolism knockdown has an effect on surviving insecticides with a broad range in mode of action. Symptoms that occur in Drosophila during exposure to the different insecticides were also noted. To gain further understanding into the mechanisms affecting survival, tissue specific knockdown was performed revealing tissue and gender specific changes in survival when vermilion, cinnabar and CG6950 are knocked down. Metabolomics was performed on the knockdown strains to determine the efficacy of the knockdowns on tryptophan catabolism and to identify any knock-on effects. The results indicate that tryptophan metabolite induced perturbations to energy metabolism and glycosylation also occur in Drosophila along with apparent changes in the absorption of ectometabolites. As the knockdown of vermilion, cinnabar and CG6950 tended to result in reduced susceptibility to insecticides, they would make poor targets for insecticidal compounds, however, they may be the first examples of genes that are not directly involved in insecticide metabolism or cuticle synthesis that increase insecticide tolerance in Drosophila. As the first metabolomics data set showed evidence for oxidative stress during permethrin exposure, preliminary work was begun for identifying the tissue specificity and timing of oxidative stress in both Dipterans and Lepidopterans using Drosophila and Bombyx mori as models. In Drosophila oxidative stress did not begin immediately suggesting that the insecticide itself is not a cause, however, a rapid increase in oxidative stress occured over a six hour period after a day of oral exposure implicating catabolites of permethrin. Bombyx were highly susceptible to permethrin showing oxidative stress in the Malpighian tubule and silk gland when exposed. This study has shown that metabolomics is highly effective at identifying pathways which modulate survival to insecticide exposure. It has also brought insight into how insecticide induced pathology may cause death. Data has also been generated which could help characterize the putative transaminase CG6950.

Single cell metabolomics provides new insights into understanding cellular heterogeneity of small molecules, and individual cell response to environmental perturbations. With high sensitivity and specificity, mass spectrometry (MS) has become an important tool for analyzing metabolites, lipids, and peptides in individual cells. Facing significant challenges, single cell and subcellular analysis by MS requires technical advances to answer fundamental biological questions, for example the phenotypic variations of genetically identical cells. The work presented in this dissertation describes my efforts to develop and apply capillary microsampling MS with ion mobility separation (IMS) for the analysis of single cells and subcellular compartments.

Chapter 1 introduces MS based analytical techniques for single cell and subcellular analysis. Recent advances of sampling and ionization methods for MS analysis of volume-limited samples are reviewed with emphasis on ambient ionization techniques, cell micromanipulation methods, and rapid gas phase separations.

In Chapter 2, the application of capillary microsampling electrospray ionization (ESI)-IMS-MS for metabolic and lipidomic analysis of single Arabidopsis thaliana epidermal cells is presented. Distinct metabolite compositions and metabolic pathways are identified among basal and pavement cells, and trichomes. These three specialized epidermal cells serve different functions in the plant leaf, and our single cell MS data reveals the corresponding metabolic pathways.

In Chapter 3, it describes the utilization of capillary microsampling ESI-IMS-MS for the analysis of metabolites and lipids in single human hepatocellular carcinoma cells. Cellular physiological states and their heterogeneity in response to xenobiotics treatment, and lipid turnover rates are explored. Here, IMS helps to enhance molecular coverage, facilitate metabolite and lipid identification, resolve isobaric ions, and minimize background interference. Comparing cells affected by metabolic modulators to unaffected counterparts reveals dramatic reduction in the availability of energy in the former.

In Chapter 4, the combination of fluorescence microscopy with capillary microsampling ESI-IMS-MS for selective analysis of identified cell subpopulations at a single cell level is demonstrated. Molecular differences and heterogeneity corresponding to cells in distinct mitotic stages are explored. Pairwise correlations between relative metabolite levels among individual mitotic cells are also studied.

In Chapter 5, the subcellular distributions of neuropeptides in individual identified neurons are explored by capillary microsampling ESI-IMS-MS. Distinct peptide distributions between the cytoplasm and nucleus are revealed. Mass spectra provide direct evidence for high abundance of these peptides in the nucleus despite the scarcity of immunostaining results supporting their presence there. A new neuropeptide is discovered and sequenced by MS in a single cell.

In Chapter 6, the current state of single cell and subcellular metabolomics is discussed. Major challenges include the low-throughput of current sampling techniques, low molecular coverage of metabolites, lipids and peptides, and external perturbations introduced by the sampling and ionization processes. In addition to exploring new solutions to these challenges, future advances will lead to the development of systems biology at the single cell level, to nano- and micro-fabricated tools to study perturbations in a lab-in-a-cell framework, and to coupling with optical manipulations and microfluidic techniques to investigate subcellular heterogeneity.

The stream of new technological advancements and their integration into the field of microbiology have contributed significantly towards our understanding of life in the micro-scale world, making the fields of microbiology and biotechnology shine like never before. Since 1980, the recombinant protein-based therapeutics industry has become one of the fastest growing sectors in the biopharmaceutical market. Nearly 30% of commercially available recombinant proteins are produced in Escherichia coli, making this species one of the most commonly used bacterial expression systems for the production of recombinant biotherapeutics. However, when it comes to the production of enzymes and bioactive secondary metabolites (antibiotic, antifungal, antiviral and immunosuppressant), Streptomyces species remain the major producer within this sector. Meeting the high demand for such products requires a clear and in-depth understanding of the bioprocesses involved to achieve high yield and quality products, whilst keeping the process industrially attractive. It is generally accepted that the metabolome, as a down-stream process to the genome and proteome, may provide a clearer picture of a biological system. Thus, in this thesis a series of metabolomics approaches were adopted to obtain a deeper insight into the metabolic effects of recombinant protein production in E. coli and Streptomyces lividans. Furthermore, a Geobacter-based biomagnetite nanoparticle production system which displayed a prolonged lag phase upon scale-up was investigated by employing metabolic profiling and fingerprinting approaches combined with multivariate analysis strategies, to identify growth-limiting metabolites. The results of this analysis identified nicotinamide as the growth limiting metabolite. Nicotinamide-feeding experiments confirmed the above findings, leading to improved biomass yield whilst restoring the lag phase to bench-scale level. Raman and Fourier transform infrared spectroscopies combined with stable isotopic probing strategies were also employed to demonstrate the application of metabolic fingerprinting in providing detailed biochemical information for quantitative characterisation and differentiation of E. coli cells at community and single-cell levels. The single-cell approach proved promising, offering detailed biochemical information and perhaps accompanying other cultivation-free approaches such as metagenomics for further future investigations. It is hoped that the advances made in these studies have proved the potential applications of metabolomics strategies to aid the optimisation of microbially-driven bioprocesses.

The leishmaniases are neglected tropical diseases with an urgent need for effective drugs. Better understanding of the metabolism of the causative parasites will hopefully lead to development of new compounds targeted at critical points of the parasite’s biochemical pathways. In my work I focused on the pentose phosphate pathway of Leishmania, specifically on transketolase, sugar utilisation, and comparison between insect and mammalian infective stages of the parasites. The pentose phosphate pathway (PPP) is the major cellular source of NADPH, an agent critical for oxidative stress defence. The PPP uses glucose, reduces the NADP+ cofactor and produces various sugar phosphates by mutual interconversions. One of the enzymes involved in this latter part is transketolase (TKT). A Leishmania mexicana cell line deleted in transketolase (Δtkt) was assessed regarding viability, sensitivity to a range of drugs, changes in metabolism, and infectivity. The Δtkt cell line had no obvious growth defect in the promastigote stage, but it was more sensitive to an oxidative stress inducing agent and most of the drugs tested. Most importantly, the Δtkt cells were not infective to mice, establishing TKT as a new potential drug target. Metabolomic analyses revealed multiple changes as a consequence of TKT deletion. Levels of the PPP intermediates upstream of TKT increased substantially, and were diverted into additional reactions. The perturbation triggered further changes in metabolism, resembling the ‘stringent metabolic response’ of amastigotes. The Δtkt cells consumed less glucose and glycolytic intermediates were decreased indicating a decrease in flux, and metabolic end products were diminished in production. The decrease in glycolysis was possibly caused by inhibition of fructose-1,6-bisphosphate aldolase by accumulation of the PPP intermediates 6-phosphogluconate and ribose 5-phosphate. The TCA cycle was fuelled by alternative carbon sources, most likely amino acids, instead of glucose. It remains unclear why deletion of TKT is lethal for amastigotes, increased sensitivity to oxidative stress or drop in mannogen levels may contribute, but no definite conclusions can be made. TKT localisation indicated interesting trends too. The WT enzyme is present in the cytosol and glycosomes, whereas a mutant version, truncated by ten amino acids, but retaining a C-terminal targeting sequence, localised solely to glycosomes. Surprisingly, cells expressing purely cytosolic or glycosomal TKT did not have different phenotypes regarding growth, oxidative stress sensitivity or any detected changes in metabolism. Hence, control of the subcellular localisation remains unclear as well as its function. However, these data are in agreement with the presumed semipermeable nature of the glycosome. Further, L. mexicana promastigote cultures were grown in media with different combinations of labelled glucose and ribose and their incorporation into metabolism was followed. Glucose was the preferred carbon source, but when not available, it could be fully replaced with ribose. I also compared metabolic profiles from splenic amastigotes, axenic amastigotes and promastigotes of L. donovani. Metabolomic analysis revealed a substantial drop in amino acids and other indications coherent with a stringent metabolic response in amastigotes. Despite some notable differences, axenic and splenic amastigotes demonstrated fairly similar results both regarding the total metabolic profile and specific metabolites of interest.

This thesis examines the relationship between insect life history and behavioural decisions and underlying cellular biochemistry, with particular focus on bethylid parasitoid wasps in the genus Goniozus. This comprises the first major body of work attempting to draw links between the underlying metabolome of an organism and its behaviour. This thesis further optimised the first known example of a combined LC-MS and NMR metabolomic approach capable of analysing extremely low biomass samples (<1 mg), a vital requirement when studying the behaviour of individual organisms. Part 1 of this thesis details the optimisation and validation of this metabolomic approach, whilst also examining the effects of aging on the metabolome of adult Goniozus wasps. Part 2 applies this approach to examine the effects of diet, host species and host aging on Goniozus wasp behaviour and biochemistry. Comparisons of the metabolomes of starved and honey fed wasps indicate that G. legneri is capable of utilising a carbohydrate rich diet as an energy source. Aged honey fed wasps possessed higher levels of large storage lipids, such as tri- and diacylglycerides, than starved wasps of the same age. Metabolomic analysis also detected a legacy effect on the metabolome of G. legneri associated with differences in the species of host each wasp was reared on. A similar legacy effect was confirmed when examining the metabolomes of wasps reared on artificially aged hosts. Whilst Goniozus wasp oviposition behaviour was altered by the species of host presented, no links between changes in a wasp’s metabolome and its resulting contest behaviour were found. Part 3 of this thesis examines the morphological, behavioural and chemical mimicry of another wasp, the hyperparasitoid Gelis agilis. G. agilis demonstrated an enhanced predation avoidance rate compared with control species, similar to that of the black garden ant Lasius niger. Agitation of G. agilis also resulted in the chemical emission of a known ant alarm pheromone.

Visceral Leishmaniasis (VL) is a major health problem worldwide and both, drugs have toxic effects and parasites developed resistance therefore new compounds and drug targets are sought. In this thesis Leishmania (L) and leishmaniasis were probed both in biochemical and clinical studies. The 6-aminonicotinamide effect was analysed on L parasite growth and metabolism by metabolomics technology. The rational was pentose phosphate pathway (PPP), a major antioxidants provider, has been reported as a 6-AN target and L PPP enzymes have significant differences versus those of the mammals, thus PPP might represent a good target. The other biochemical study was conducted to characterize L transglutaminase (TGase), catalyzing irreversible protein crosslinking; if fundamental for L parasites, these enzymes could represent another promising drug target. In the thesis clinical part, subclinical L infections were evaluated in autoimmune rheumatic patients treated with immunosuppressive drugs at Ferrara Hospital in Italy. L. mexicana and L. infantum promastigotes were treated with 7.8 mM 6-AN for 24 hours and small metabolites were extracted and analysed by pHILIC-LC-MS. TGase was tested in vivo in canine L. infantum promastigotes as well as in vitro in parasite extracts using fluorescein-cadaverine. Transamidation in AS precipitated fractions was measured in microwell plates. Western blot of human L. infantum lysate and canine L. infantum extract and precipitated fractions as well as immunocytochemical analysis of canine L. infantum were performed using human TGase 2 polyclonal antibodies. L PCR and real-time PCR were performed on DNA extracted from PBMCs from 50 autoimmune rheumatic patients and 50 healthy subjects. Plasma cytokines were also measured. In both L. mexicana and L. infantum, 6-AN caused significant depletion of phosphoribosylpyrophosphate (PRPP) and nicotinate and as a result purine and pyrimidine nucleotides were reduced and their nucleobases accumulated. Glutathione, ribose-5-phosphate, 6-phosphogluconate levels and downstream PPP intermediates were similar to controls. In mammals 6-AN is converted to abnormal 6-ANAD/P by NAD+ glycohydrolase, however, in L its toxicity is only seen in mM range, in which 6-AN depletes the cellular PRPP content probably in the Preiss-Handler NAD+ salvage pathway, resulting in depletion of nucleotides required for nucleic acid biosynthesis. L NAD+ glycohydrolase might decompose NAD+ but not catalyze exchange reactions as found in other microrganisms, however, combined 13C-glucose labeling and flux analysis might be useful to ascertain the fate and action mechanism of 6-AN in L. TGase was detected and confirmed in vivo and/or in vitro in human and canine L. infantum promastigotes. The activity in the canine strain was found Ca2+-dependent and inhibited by 10mM GTP. Immunocytochemistry showed fluorescent protein and blots revealed a band of 74.6 KDa for the canine strain and two bands of 55.34 KDa and 65.87 KDa for the human strain, thus the TGase pAbs (orb2986) could permit purification of this L enzyme. Eighteen autoimmune rheumatic patients were positive for L DNA by PCR and/or quantitative PCR. No statistical difference observed in relation to ownership of a dog or the type of biological drug used. Pro-inflammatory IL-1, IL-6, IL-12(p70), IL-7, IL-15, IFN-γ and TNF-α; anti-inflammatory IL-4, IL-13; and regulatory IL-10 cytokines were markedly elevated in patients with additional increase those positive for L DNA. The high L parasitaemia detected suggests that biologic treatment can lead to cryptic VL or L infection in a latent phase which may progress to full VL in the setting of immunosuppression. Molecular screening for L using PBMC fractions and cytokine analysis should be performed before treating autoimmune rheumatic patients with biologic drugs.
La leishmaniosi viscerale (VL) è un problema sanitario mondiale e i farmaci possono dare tossicità nonché generare resistenza, perciò è importante la ricerca di nuovi composti e nuovi “drug target”. In questa tesi la Leishmania (L) è stata studiata sia dal punto di vista biochimico che clinico. Ho analizzato gli effetti antiproliferativi e metabolici della 6-aminonicotinamide. Essendo riportato come bersaglio principale della 6-AN la via dei pentosi fosfato (PPP), necessaria al sistema di difesa antiossidante della cellula e i cui enzimi in questi parassiti presentano differenze significative con quelli di mammifero, PPP potrebbe rappresentare un buon ”drug target”. Inoltre, poiché un secondo bersaglio potrebbe essere la transglutaminasi (TGasi) riportata essere importante per il parassita, si è cercato di caratterizzarla in L. infantum. Nello studio clinico, si è valutata la prevalenza di parassitosi da L, asintomatiche/subcliniche in pazienti dell’ospedale di Ferrara, affetti da patologie reumatiche autoimmuni, trattati con farmaci immunosoppressori. Promastigoti di L. mexicana e L. infantum sono stati trattati per 24 ore con 6-AN 7,8 mM e dopo estrazione i metaboliti più piccoli sono stati analizzati mediante pHILIC-LC-MS. La TGasi è stata saggiata in vivo in promastigoti di L. infantum e in vitro in lisato cellulare dopo incubazione con fluoresceina-cadaverina. L’attività è stata anche saggiata in micropiastra pure dopo frazionamento con AS. Anticorpi policlonali contro TGasi 2 umana sono stati usati nell’immunocitochimica e in Western Blot di lisati di L. infantum umana e canina e frazioni precipitate. Su DNA estratti da PBMC di 50 pazienti e 50 soggetti sani si sono effettuate PCR qualitativa e real-time. Inoltre si sono quantificate le citochine seriche. Sia in L. mexicana che L. infantum, 6-AN ha causato una diminuzione significativa di fosforibosil-pirofosfato (PRPP) e nicotinato e come conseguenza di nucleotidi mentre le nucleobasi sono aumentate. I livelli di glutatione, ribosio-5-fosfato, 6-fosfogluconato e intermedi PPP a valle erano simili ai controlli. Nei mammiferi la 6-AN è metabolizzata a 6-ANAD/P da una NAD+ glicoidrolasi, ma in L la tossicità solo in concentrazione mM, con deplezione di PRPP e conseguente calo di nucleotidi, indica che 6-AN potrebbe entrare nella via Preiss-Handler di sintesi del NAD+. In L la NAD+ glicoidrolasi potrebbe idrolizzare il NAD+ ma non catalizzare lo scambio tra nicotinamide e 6-AN, come riportato in altri microrganismi. Analisi ulteriori di flusso dopo marcatura con 13C-glucosio potrebbero chiarire il meccanismo d’azione del 6-AN in L. Si è confermata in vivo and in vitro, la presenza in L di una TGasi attiva, in promastigoti canini Ca2+-dipendente e inibita da GTP. Con l’immunocitochimica si è rivelata fluorescenza e nei blot bande di 74.6 KDa per il ceppo canino e di 55.34 e 65.87 KDa per quello umano, suggerendo che gli anticorpi usati potrebbero essere utili nella purificazione dell’enzima. In 18 pazienti si è riscontrata positività ma non evidenziate differenze significative tra possessori o meno di cani e in riferimento al tipo di farmaco somministrato. In tutti i pazienti in trattamento con farmaci biologici, erano significativamente elevate le citochine pro-infiammatorie IL-1, IL-6, IL-12(p70), IL-7, IL-15, IFN-γ e TNF-α, quelle anti- infiammatorie IL-4 e IL-13 e la regolatoria IL-10, ma un aumento maggiore era presente nei pazienti positivi per la presenza di DNA di L. L’alta parassitemia trovata suggerisce che il trattamento possa portare a VL criptica o infezione latente, con possibilità di progressione a VL conclamata in caso di evoluzione a uno stato di immunocompromissione. Si potrebbero quindi introdurre uno screening molecolare su frazioni PBMC e l’analisi di citochine prima di prescrivere questo tipo di farmaci a pazienti con patologie autoimmuni.

Metabolomics is defined as the systematic analysis of hundreds or thousands of small metabolites present in a living system. It has emerge as an important field of study along with other, already established ‘omics’ sciences, i.e., genomics, proteomics and transcriptomics. Metabolomics is well established in the field of medicine, drug toxicity and disease diagnosis. Among the existing analytical techniques, NMR is a fast, reproducible and non-destructive technique to construct an informative snapshot of the metabolites under certain conditions. NMR data give metabolic signature information of the samples when it is combined with data preprocessing and chemometric tools, such as multivariate statistical techniques. NMR-based metabolomics is still expanding in the field of the food chemistry. In this context, this Ph.D. thesis is focused on two major aspects, which show applications of NMR-based metabolomics in food chemistry. 1. Many nutraceutical products possess powerful antioxidant activity as demonstrated in many chemical in vitro tests and in several in vivo trials. Nevertheless, the mechanism of their activity is not completely studied in detail. Due to their poor bioavailability and fast metabolism, studies on the in vivo antioxidant effects are still needed. We performed longitudinal experiments on Sprague Dawley (SD) rats using two commonly available nutraceutical antioxidant products, namely, Curcumin (chapter 2) and Resveratrol (chapter 3). The effects of different doses of orally administered standardized antioxidant extracts in healthy rats were investigated by untargeted metabolomic analysis based on LC-MS and NMR spectrometry. The experiments were carried out over different periods of time for different antioxidants. Changes in the urinary metabolome were evaluated by monitoring the 24-hour urine composition by 1H-NMR and HPLC-MS. The two different approaches were able to detect variations in the urinary levels of antioxidant markers, leading to the observation of different metabolites thus proving the complementarity of these two analytical techniques for metabolomic purposes. Analytical tools such as MS and NMR spectroscopy in combination with chemometrics can profile the impact of time, stress, nutritional status, and environmental perturbations on hundreds of metabolites simultaneously. This results in complex, massive data sets that must be analyzed through a careful statistical protocol. Our strategy included data preprocessing, data analysis and validation of statistical models. After several data processing steps, principal component analysis (PCA) and partial least-squares (PLS) were used to identify urine biomarkers. The PLS models were validated by permutation tests and critically important variables were validated through univariate analysis. 2. The second part of this thesis (chapters 4 and 5) describe the use of NMR-based metabolomics as a fast, convenient, and effective tool for origin discrimination and biomarker discovery in food analysis. Traditionally, the determination of the floral origin of honey is made from palynological analysis. The method is based on the identification of pollen by microscopic inspection. However, melissopalynological analysis needs expertise and also it is not a very reliable technique for the discrimination of botanical origin of some types of honey. Also, honey regulation in the EU (Codex Alimentarius Commission 2001; European Commission 2002) emphasizes that the botanical and geographical origins of the product must be printed on the label in order to avoid the fraud such as adulteration with industrial sugar, selling product under false name or mixing the honey of different floral origin. Therefore, there is need to establish a method to discriminate honey from different origin. The aim of this work was to develop an NMR-based metabolomic approach that used multivariate statistical analysis to discriminate the botanical (chapter 4) and entomological (chapter 5) origin of different types of honey. Multivariate statistical analysis helped us to identify the most relevant signals to differentiate honey botanically and entomologically. The obtained data sets were useful in the search of markers responsible for the discrimination of different honey samples from different botanical species and produced by different bee species.
La metabolomica è definita come l'analisi sistematica di centinaia o migliaia di piccoli metaboliti presenti in un sistema vivente. È emerso come un importante campo di studio insieme ad altre, già affermate scienze "omiche", vale a dire, genomica, proteomica e trascrittomica. La metabolomica è ben consolidata nel campo della medicina, nello studio della tossicità di farmaci e nella diagnostica. Tra le tecniche analitiche esistenti, NMR è veloce, riproducibile e non distruttiva, utile per fornire una fotografia informativa sui metaboliti in determinate condizioni. Dati NMR forniscono informazioni metaboliche che caratterizzano i campioni quando combinati con una pre-elaborazione dei dati e con strumenti chemiometrici, come le tecniche di statistica multivariata. La metabolomica basata sull’NMR è ancora in espansione nel campo della chimica degli alimenti. In questo contesto, questa tesi di Dottorato si concentra su due aspetti principali, che mostrano applicazioni della metabolomica basata sull’NMR in chimica degli alimenti. 1. Molti prodotti nutraceutici possiedono potente attività antiossidante, come dimostrato in molti test chimici in vitro e in diverse prove in vivo. Tuttavia, il meccanismo della loro attività non è completamente studiato in dettaglio. A causa della loro scarsa biodisponibilità e metabolismo veloce, sono ancora necessari studi in vivo sugli effetti antiossidanti. Abbiamo condotto esperimenti longitudinali su ratti Sprague Dawley (SD) utilizzando due prodotti antiossidanti nutraceutici comunemente disponibili, vale a dire, curcumina (capitolo 2) e resveratrolo (capitolo 3). Gli effetti di diverse dosi di estratti antiossidanti standardizzati somministrati per via orale nei ratti sani sono stati studiati mediante analisi metabolomica non mirata (untargeted) basata su LC-MS e spettrometria NMR. Gli esperimenti sono stati eseguiti lungo diversi periodi di tempo per diversi antiossidanti. Le variazioni del metaboloma urinario sono state valutate attraverso il monitoraggio della composizione delle urine di 24 ore usando 1H-NMR e HPLC-MS. I due differenti approcci sono stati in grado di rilevare le variazioni dei livelli urinari di marcatori antiossidanti, portando all’osservazione di diversi metaboliti e dimostrando così la complementarità di queste due tecniche analitiche per scopi metabolomici. Strumenti di analisi come la spettroscopia NMR e MS in combinazione con chemiometria possono delineare l'impatto del tempo, dello stress, dello stato nutrizionale, e di perturbazioni ambientali su centinaia di metaboliti contemporaneamente. Ciò comporta complessi enormi set di dati che devono essere analizzati mediante un protocollo statistico accurato. La nostra strategia ha compreso una pre-elaborazione dei dati, l’analisi dei dati e la validazione dei modelli statistici. Dopo varie fasi di elaborazione di dati, l’analisi delle componenti principali (PCA) e l’analisi dei minimi quadrati parziali (PLS) sono state utilizzate per identificare i biomarcatori urinari. I modelli PLS sono stati convalidati dai test di permutazione e le variabili di importanza critica sono stati convalidati attraverso analisi univariata. 2. La seconda parte di questa tesi (capitoli 4 e 5) descrivono l'uso di metabolomica basata su NMR come strumento veloce, conveniente ed efficace per la discriminazione di origine e la scoperta di biomarcatori in analisi degli alimenti. Tradizionalmente, la determinazione dell'origine floreale del miele è condotta mediante analisi palinologica. Il metodo si basa sulla individuazione di polline mediante ispezione microscopica. Tuttavia, l'analisi melissopalinologica richiede perizia ed inoltre non è una tecnica molto affidabile per la discriminazione di origine botanica di alcuni titpi di miele. Inoltre, la regolamentazione del miele nell'Unione Europea (Codex Alimentarius 2001; Commissione Europea 2002) sottolinea che le origini botaniche e geografiche del prodotto devono essere stampate sull'etichetta, per evitare frodi, come l'adulterazione con zucchero industriale, vendita di prodotti sotto falso nome o aggiunte di miele di diversa origine floreale. Pertanto, vi è la necessità di stabilire un metodo per distinguere miele di diverse origini. Lo scopo di questo lavoro è stato quello di sviluppare un approccio metabolomico basato sull’NMR che ha utilizzato l'analisi statistica multivariata per discriminare l'origine botanica (capitolo 4) ed entomologica (capitolo 5) di diversi tipi di miele. statistica multivariata ci ha aiutato ad identificare i segnali più importanti per differenziare il miele sia dal punto di vista botanico che entomologico. I set di dati ottenuti sono stati utili nella ricerca di marcatori responsabili della discriminazione dei diversi campioni di miele di diverse specie botaniche e prodotti da diverse specie di api.

Epigenetics and metabolomics are rapidly growing areas of research, in part due to recent advances in technology that have allowed for a wide coverage of the human genome. Metabolites are small compounds present in cell and body fluids, and are involved in biochemical processes of the cell. Quantitative trait loci associated with levels of individual metabolites (mQTLs) have been identified from numerous metabolome GWAS. Here, I analysed metabolite levels in twins with the aim of identifying genetic variants that influence metabolomic traits (mQTLs) using two different metabolomics platforms, and consequently compared the results to report stable metabolites on both technologies to ultimately enable combining metabolite profiles across these two platforms. DNA methylation is a biochemical process that is vital for mammalian development. It is present throughout the genome and is the most extensively studied epigenetic mark. Previous studies have explored the heritability of DNA methylation and have identified methylation QTLs (meQTL). Here, I identified meQTLs with the goal of assesing the evidence of genetic effects influence not only DNA methylation levels, but also variability by using MZ-twin discordance as a measure of variance. Epigenetic mechanisms and metabolomic profiles have both been shown to play a role in gene expression and susceptibility for complex human disease. Here, I analysed the association between type 2 diabetes and metabolomic and epigenetic datasets and combined the data to identify connections between these levels of biological data at genetic variants linked to type 2 diabetes to gain more insight into the disease susceptibility and progression. Overall, the results confirmed previous findings of strong genetic influences on metabolites and extend current knowledge about genetic effects underlying several biochemical pathways. Additionally, the results also showed genetic influences on DNA methylation, and give insights into mechanisms by which genetic impacts epigenetic processes. Lastly, the findings show that specific genetic susceptibility variants for type 2 diabetes can also impact epigenetic and metabolomics profiles, and can help improve our understanding of the disease etiology.

There is a long history of use and modern commercial importance of large (Vaccinium macrocarpon Aiton) and small cranberries (V. oxycoccus L. and V. vitis-idaea L.) in North America. While epidemiological research indicates cranberries have positive health benefits, identifying specific phytochemicals for disease prevention remains elusive. The central objective of this research was to develop phytochemical characterization tools for comparing commercially cultivated cranberries and two wild-harvested Vaccinium species. A method was developed and validated to quantify cyanidin-3-O-galactoside (C3Ga), cyanidin-3-O-glucoside (C3Gl), cyanidin-3-O-arabinoside (C3Ar), peonidin-3-O-galactoside (P3Ga) and peonidin-3-O-arabinoside (P3Ar) in cranberry fruit products. The relative standard deviation (%RSDr) of anthocyanins ranged from 1.77% to 3.31% with the method detection limit (MDL) for C3Ga, C3Gl, C3Ar, P3Ga, and P3Ar estimated at 0.018, 0.016, 0.006, 0.013, and 0.011 µg/mL, respectively. In biological replicates of V. macrocarpon cultivars, anthocyanin content (mg/g) was determined to be 7.98±5.83 in Ben Lear; 7.02±1.75 in Bergman; 6.05± 2.51 in GH1; 3.28± 1.88 in Pilgrim and 2.81 ±0.81 in Stevens. V. oxycoccus contained the five major anthocyanins with the ratio of glycosylated peonidins to cyanidins varying from V. macrocarpon. V. vitis-idaea contained no measurable glycosylated peonidins. Determination and statistical analysis of serotonin, melatonin and ascorbic acid content in the three Vaccinium species, found only vitamin C positively correlated with antioxidant activity. Metabolome comparisons made from untargeted metabolomics by ultra-fast liquid chromatography (UFLC) with time-of-flight (TOF) mass spectrometry (MS), found different pools of common metabolites extracted in methanol (7635), 70% ethanol (5832), and water (5664). Each cranberry matrix contained unique compounds; fruit=3680, product 1=3464, product 2=3500. Clustering was visualized using principal component and partial least squares discriminate analyses with application of univariate statistics to mitigate false discoveries. A significance algorithm found 1987, 716 and 1556 compounds (p-value<0.05) in methanol, 70% ethanol and water extracts, respectively. In metabolomic profiles of V. macrocarpon varietals 6481 compounds were conserved and 136 (Ben Lear), 84 (Bergman), 91 (GH1), 128 (Pilgrim) and 165 (Stevens) unique observations were made. Across the three Vaccinium species 8000-10,000 phytochemicals were detected with 4624 compounds conserved between them. Together these data establish targeted and untargeted methods for phytochemical characterization of cranberries, providing foundational chemotaxonomic knowledge and new insights into the maintenance of health in traditional North American diets.

In dieser Doktorarbeit werden drei Metabolomics-Studien der KORA Kohorte behandelt. Das Ziel dieser Doktorarbeit war, ein besseres Verständnis der Rolle des Metabolismus von komplexen Phänotypen anhand von Unterschieden im Blutbild, des Geschlechts und anhand von Veränderungen des Metabolitenprofils bei multifaktoriellen Krankheiten wie Typ 2 Diabetes mellitus zu erhalten. Um Artefakte auszuschließen wurden strikte Qualitätskontrollen aller gemessenen Metaboliten durchgeführt. Durch die Analyse von Blutplasma und -serum von 377 Personen konnten wir zeigen, dass die Konzentrationen der Metaboliten in Blutplasma und -serum stark korrelieren und darüber hinaus eine hohe Reproduktionsrate zeigen, bei der Blutplasma besser abschneidet. Im Gegensatz dazu zeigt das Blutserum höhere Metabolitenkonzentrationen und könnte deswegen besser für den Nachweis von Konzentrationsunterschieden geeignet sein. Ein weiteres Ergebnis dieser Doktorarbeit war der Nachweis von signifikanten geschlechtsspezifischen Unterschieden der Konzentrationen von 102 der ausgewerteten 131 Metaboliten. Dabei wurden die Daten von mehr als 3300 Personen der KORA Kohorte verwendet und die Analysen einer konservativen Bonferroni-Korrektur unterzogen. Darüber hinaus identifizierten wir potentielle Biomarker für Prä-Diabetes durch die Analyse von 140 Metaboliten in nüchtern abgegebenen Blutseren von 4297 Personen der KORA Kohorte. Wir konnten zeigen, dass Personen mit gestörter Glukosetoleranz (IGT) signifikant unterschiedliche Konzentrationen von drei Metaboliten (Glycin, lysoPhosphatidylcholine (LPC) 18:2 und acetylcarnitine) im Vergleich zu gesunden Personen aufweisen. Darüber hinaus konnten wir nachweisen, dass geringere Konzentrationen der Metaboliten Glycin und LPC bei Probanden mit Typ 2 Diabetes oder IGT vorhanden sind. Die in dieser Studie identifizierten Metaboliten sind biologisch unabhängig von zuvor entdeckten Diabetes Risikofaktoren. Durch weitere Analysen und die Einbeziehung systembiologischer Ansätze entdeckten wir sieben Diabetesrisiko Susseptibilitätsgene, welche durch Expressionsdaten bestätigt wurden. Metabolomics welches auf der Analyse von Stoffwechselzwischen- und Endprodukten basiert, ist eine wertvolle Methode besonders in der biomedizinischen Forschung, um Krankheitsmechanismen aufzuklären. Nachdem angemessene Qualitätskontrollen etabliert und der Einfluss von komplexen Störfaktoren (z.B. das Geschlecht) aufgeklärt wurden, konnte der Zusammenhang zwischen Krankheit und Metabolismus weiter an Klarheit gewinnen. Die Entdeckungen in unserer T2D Studie zeigen, dass die Analyse von Konzentrationsprofilen helfen kann neue Krankheitsrisikomarker genauso wie neue Wirkungspfade zu identifizieren, die möglicherweise das Ziel zur Heilung einer Krankheit sein könnten.

Includes bibliographical references
Resurrection plants are unique in the ability to survive near complete water loss in vegetative tissues without loss of viability. In order to do so, they employ multifaceted strategies which include structural adaptations, antioxidant and photoprotective mechanisms, and the accumulation of proteins and metabolites that stabilise macromolecules. A full understanding of the phenomenon of vegetative desiccation tolerance will require a systems view of these adaptations at the levels of the genome, the control of gene expression, and the control of metabolic pathways. This dissertation reports a high-throughput metabolomic analysis of the changes that occur in vegetative tissues of resurrection plant Xerophyta humilis during dehydration. A combination of chromatography, mass spectrometry and nuclear magnetic resonance revealed numerous primary and secondary metabolites in the plant. Multivariate statistics identified a subset of metabolites that were significantly up- or down-regulated in response to water deficit stress. These metabolites both confirmed existing observations about the metabolic response of X. humilis to drying and revealed compounds not previously known to be associated with this response. Desiccation-associated metabolites were mapped onto known biochemical pathways, to generate hypotheses concerning possible regulatory schemes in the stress response, inviting deeper investigation in future.

Alzheimer's disease (AD) is a major debilitating disease with both cognition and independence gradually lost over time. AD can be diagnosed post-mortem when the accumulation of amyloid-β (Aβ) peptides and tau proteins in the brain are visible. The best diagnostic markers at present are cerebrospinal fluid (CSF) Aβ and tau levels. However, the diagnostic accuracies of CSF markers are limited and all clinical trials targeting Aβ and tau pathologies have failed to show promising results. Therefore, there is an urgent need for new molecular leads associated with AD which can potentially be utilised as disease-modifying therapy agents and as AD biomarkers. This thesis investigates the possibility of using metabolomics to discover AD associated metabolites. Initially in chapter two, CSF samples (20 AD, 20 age-matched healthy controls) were analysed by Nuclear Magnetic Resonance (NMR). Results showed that metabolic fingerprints were non-differentiable between the two cohorts. One metabolite was found decreased, pyruvate, suggesting energetic hypometabolism in AD (p < 0.05). Acetate levels were increased in the AD group (p < 0.05), suggesting acetate being used as an alternative energy source to pyruvate. In the next chapter, in order to scan for more metabolites, Liquid Chromatography- Mass Spectrometry (LC-MS) was applied on the same cohorts. The experiments allowed for the detection of 4426 metabolic features. Although the fingerprints were non-differentiable between the cohorts, 2 unidentified metabolic features were found to be able to discriminate AD from age-matched controls with a Receiver Operating Characteristic (ROC) Area Under the Curve (AUC) value of 70%. After this, work focused in blood metabolites because they are easily accessible. In chapter four we wanted to test whether lipids that had been previously implicated with AD would reproduce with similar trends. Semi-targeted analysis of 9 lipids was carried out (3 phosphatidylcholines (PC) and 6 ceramides) in AD (n=205) and Controls (n=207). Elevated levels of three ceramides and diminished levels of 2 PC molecules were found in AD blood (p < 0.05), one PC associated with hippocampal atrophy. In the following chapter, these 9 lipids along with cholesterol and absolute cholesteryl esters levels were measured on pre-conversion serum samples (112 eventual AD participants and 113 Control participants at 3 time-points). At timepoints 1 and 2 (pre-clinical stage), no lipids showed significant differences between the pre-converters and the stable cohorts. One PC and total cholesteryl ester levels were diminished at the symptomatic stage, time point 3 (p < 0.01). When using lipidomics, the whole fingerprints and 1207 metabolic features, one feature (annotated as a PC) was found to be significantly different at symptomatic stage (q < 0.05). Finally in chapter six, untargeted lipidomic analysis was performed on 148 AD and 152 control plasma samples in search of AD blood biomarkers. Application of random forest showed a combination of 24 lipid features consisting of cholesteryl ester, triglycerides and phosphatdiylcholines being able to discriminate the cohorts with AD classification accuracy of > 70%. In addition, other lipid signatures were found to predict disease progression (R2=0.10) and brain atrophy in all brain regions except for left entorhinal cortex (R2 > 0.14).

Inflammatory bowel disease represents a group of chronic disorders that affect one or more parts of the intestine. Although recently the incidence of inflammatory bowel disease has noticeably increased, its aetiology is still unclear. No specific pathogen has been defined as a causative agent. Serological biomarkers have been recently proposed for diagnosis, but they remain untested in clinical applications. Moreover, current diagnostic and monitoring practices for inflammatory bowel disease are very invasive. Therefore, accurate tools for the early diagnosis, and in particular non-invasive strategies are needed. One area of recent interest is the relationship between the metabolome and microbiota in terms of inflammatory bowel disease pathogenesis and intervention strategies. Direct analysis of metabolites and their interaction with the gut microbial species is a rapidly growing field of research. In this context, metabolomics could represent a useful approach to understand possible pathological mechanisms or metabolite modification in different pathways. Metabolomics is based on the quantitative measurement of dynamic metabolic changes in living systems in response to physiological stimuli or genetic modifications, nutrients and drugs. Thanks to the comprehensive study of low molecular weight metabolites (<1500 Da) in biofluids and in tissues, metabolomics ensures the characterization of the metabolic phenotype of a living organism. In this work, the polar and the lipid metabolite profile of faecal and plasma samples of patients affected by inflammatory bowel disease and healthy patients has been studied by high resolution liquid chromatography coupled with several technological platforms such as a triple quadrupole, a quadrupole time of flight mass spectrometry and an ion mobility prior to multivariate statistical data analysis. By these means, differences between Crohn’s disease, ulcerative colitis and control samples were investigated. Results of discriminant analysis were considered with the aim to find the relevant metabolites unique for each class. The results highlight differences in the metabolite profile between the pathological and the control samples. In faecal samples, the most discriminant metabolites for both IBD classes were diacylglycerols, phosphatidylcholines triacylglycerols and tetra pyrrole compounds. Furthermore, plasma samples results showed modification of the lysophosphatidylcholine and phosphatidylcholine pathways strictly related to the arachidonic acid response. These results highlight similarity in the metabolic alteration occurring in both ulcerative colitis and Crohn’s disease when compared to healthy controls. Modification to the endocannabinoid system were also found regarding the ulcerative colitis plasma and faecal samples. All the lipid results were correlated with the targeted analysis of the polar metabolites profile. In conclusion, the application of metabolomics to faecal and plasma samples of IBD patients allows for the identification of metabolites that can be used as possible indicators of metabolic pathways implicated in the onset of these pathologies.

Tuberculosis (TB) is one of the deadliest infectious diseases of our time, with 1.4 million deaths globally, recorded in 2010 (3800 deaths a day) by the World Health Organization (WHO). Currently, South Africa ranks third on the 2011 list of 22 high-burden TB countries in the world and it was estimated that each active-TB person could potentially infect 10–15 people annually. The WHO additionally reported that in the year 2009, 87% of all TB patients worldwide were successfully treated, with a treatment success rate of 74% reported for South Africa. Despite this however, non-adherence to anti-TB treatment is still a major issue, due to it resulting in a global increased prevalence of drug resistant TB and subsequently TB treatment failure. Treatment failure is thought to be caused by a number of factors, however, it still remains largely misunderstood. One aspect of this, that isn't clearly addressed in the literature, is the underlying variation in each patient, resulting in his/her varying reaction to the drug regimen, and hence it’s varying efficacy from one patient to the next. Furthermore, little is known about the underlying variation of the host to the primary TB infection or response to the TB disease state, and how some patients have more effective mechanisms for eliminating the infection, or recovering from the disease. Considering this, a metabolomics research study using GC×GC-TOFMS was conducted, in order to identify potential metabolite markers which may be used to better characterise the underlining mechanisms associated with poor treatment outcomes (treatment failure). The first aim was to evaluate the accuracy and efficiency of the methodology used, as well as to determine the capability and accuracy of the analyst to perform these methods. In order to evaluate the GCxGC-TOFMS analytical repeatability, one QC sample was extracted and injected repeatedly (6 times) onto the GC×GC-TOFMS. Similarly, the analyst's repeatability for performing the organic acid extraction and analyses was also determined, using 10 identical QC samples, which were extracted and injected separately. CV values were subsequently calculated from the collected and processed data as a measure of this. Of all the compounds detected from the 6 QC sample repeats used for GCxGC-TOFMS repeatability, 95.59% fell below a 50% CV value, and 93,7% of all the compounds analysed for analyst repeatability had a CV < 50. Subsequently, using the above metabolomics approach, in addition to a wide variety of univariate and multivariate statistical methods, two patient outcome groups were compared. A sample group cured from TB after 6 months of treatment was compared vs a sample group where treatment failed after the 6 month period. Using urine collected from these two patient groups at various time points, the following metabolomics comparisons where made: 1) at time of diagnosis, before any anti-TB treatment was administrated, 2) during the course of treatment, in order to determine any variance in these groups due to a varying response to the anti-TB drugs, 3) over the duration of the entire 6 months treatment regimen, in order to determine if differences exist between the two groups over time. A clear natural differentiation between the cured and failed outcome groups were obtained at time of diagnosis, and a total of 39 metabolites markers were subsequently identified. These metabolites were classified according to their various origins, and included (1) those associated with the presence of M. tuberculosis bacteria, (2) those resulting from an altered host metabolism due to the TB infection, and (3) metabolites of various exogenous origins. The detailed interpretation of these metabolites suggests that a possible underlying RCD or some sort of mitochondrial dysfunction may be present in the treatment failure group, which may also be induced through an external stimulus, such as alcohol consumption. We hypothesise that this may possibly result in a far greater severity to M. tuberculosis infection in this group, subsequently causing a reduced capacity for a successful treatment outcome, also considering the critical role of the mitochondria in the metabolism of anti-TB drugs. Furthermore, 20 metabolite markers were identified when comparing the two outcome groups during the treatment phase of this metabolomics investigation. A vast majority of these 20 metabolites were also identified as markers for time 0 (time of diagnosis). Additionally, metabolites associated with anti-TB drug induced side effects, were also found to be comparatively increased in the treatment failure group, indicative of more pronounced liver damage, accompanied by metabolites characteristic of a MADD metabolite profile, due to a deficient electron transport flavoprotein, confirming previous experiments done in rats. These side effects have also previously been implicated as a major contributor of poor treatment compliance, and ultimately treatment failure. Lastly, 35 metabolite markers were identified by time dependent statistical analysis and represented those metabolites best describing the variation between the treatment outcome groups over the entire study duration (from diagnosis, to week 26). This time dependent statistical analysis identified markers, using an alternative statistical approach, and confirmed previous findings and added in a better characterisation of treatment failure. Considering the above, we successfully applied a metabolomics approach for identifying metabolites which could ultimately aid in the prediction and monitoring of treatment outcomes. This additionally led to a better understanding and or characterisation of the phenomenon known as treatment failure, as well as the underlying mechanisms related to this occurrence.
MSc (Biochemistry), North-West University, Potchefstroom Campus, 2013

Diabetic nephropathy is a chronic kidney disease and one of the more severe complications from diabetes mellitus type 2. The glomerular and tubular dysfunctions usually lead to end stage renal disease and the treatments of these patients (dialysis, kidney transplants) are a huge economic burden for the society. Due to an epidemiologic increase of type 2 diabetes, conventional diagnostic markers like creatinine and albumin are not sufficient, since they are only able to identify already existing kidney damage. With targeted metabolomics, the analysis of small molecules produced from metabolism, this project aimed at finding novel and more sensitive metabolic biomarkers from several different classes of metabolites. The different assays were performed with flow injection analysis, high performance liquid chromatography, gas chromatography and mass spectrometry, and with principal component analysis and discriminant analysis, up-and down-regulated metabolites could be identified and their respective biochemical pathways, if possible, explained. In diabetics significantly elevated concentrations of very long chain fatty acids (impaired peroxisomal β-oxidation), urinary sugars and acylcarnitines in plasma could be recognized. Markers indicating kidney damage included significantly increased plasma concentrations of asymmetric dimethylarginine (inhibition of nitric oxide synthase resulting in decreased endothelial functionality) and histamine (indication of uremic pruritus). Oxidative stress was also found to be a potential prognostic marker as indicated by the raised methionine-sulfoxide to methionine ratio in nephrotic patients. To summarize, this project succeeded in identifying metabolic biomarkers both for diabetes type 2 and nephropathy, which in the future might become important tools in slowing down progression or diagnosing these diseases.

Troglitazone was introduced as an anti-diabetic drug in the late 1990s, and represented the first of the thiazolidinedione (TZD) class of drugs to be marketed. Troglitazone works by activating the peroxisome proliferator-activated receptor y (PP ARy), reducing blood glucose levels by enhancing insulin sensitivity, primarily in the adipose and skeletal muscle tissues. Due to increasing concerns of troglitazone-linked hepatotoxicity, troglitazone was withdrawn from the U'K. market in 1997 and U.S.A. in 2000. A considerable effort to elucidate the mechanisms of troglitazone-induced hepatotoxicity has been made, resulting in a variety of proposed mechanisms; formation and accumulation of toxic metabolites, oxidative stress, mitochondrial dysfunction, and inhibition of bile salts excretory protein leading to cholestasis. The purpose of this study was to investigate whether a metabolomics approach can be used to further elucidate the mechanism of toxicity of troglitazone. Furthermore, metabolomic profiling was undertaken to determine if this approach can distinguish between TZDs with hepatotoxic potential (i.e. troglitazone) and those with none (i.e. rosiglitazone and pioglitazone). Conventional MTT assay revealed that troglitazone was a more potent toxin to the HuH-7 hepatoma cell line compared to rosiglitazone and pioglitazone, with an ICso value of 25.9 ± 1.8, 2: 168.9 ± 30.0, and 2: 79.4 ± 13.5 ~M, respectively. A comparison between the ICso values obtained by the MTT versus LDH assays (25.9 ± 1.8 versus 2: 42.7 ± 3.6 ~M) suggests that troglitazone affected the reductive capacity of mitochondria at lower level before causing the cells to rupture and release their intracellular contents. An untargeted and a targeted metabolomics approach revealed that troglitazone decreased the levels of intracellular glutamate, malate, energy production, GSH, and nicotinamide at concentrations that are non-toxic by the MTT assay. These observations demonstrate the discriminative ability of a metabolomics approach for the assessment of drug toxicity. The findings also suggest that troglitazone affects the tricarboxylic acid (TCA) cycle, interrupting the anaplerotic and cataplerotic balance of the TCA cycle, leading to oxidative stress and reduced energy level.

The aim of this study was to determine if metabolome analysis (“metabolomics”) can reveal a phenotype for S. cerevisiae strains with deleted genes that show no observable difference from wild type, with respect to growth rate on defined media. It was found that quenching cellular metabolism using ice cold water and collecting intracellular metabolites, whilst the samples were in stationary phase, gave the most reproducible profiles. Using this technique it has been shown that strains of S. cerevisiae with deleted genes in biologically distinct pathways can be distinguished. For example, strains with a gene deleted in aerobic respiration could be distinguished from profiles of strains that affect pyrimidine and proline metabolism. However a significant separation of strains whose genes affect more closely related areas of metabolism was not observed. For example proline and pyrimidine mutants could not be separated using this technique. However the technique did reveal a unique metabolite signature for one strain with a gene deletion affecting pyrimidine metabolism, which was not predicted from prior consideration of the metabolic pathway. Metabolome analysis was also applied to a more complex organism, the mouse, to determine whether genotype differences also give characteristic metabolic responses in a more complex system. Metabolite profiles from the urine of asymptomatic mice that were prone to develop Type 1 diabetes were compared with profiles in urine collected from genetically similar strains that were largely protected against the disease. The separation of animals in accordance with their genetic differences was observed. For example mouse strains which differ at only one genetic locus could be distinguished with this technique. However distinguishing strains according to their propensity to diabetes revealed a weak association.

Alcohol is a substance used and abused by many individuals. The metabolic perturbations caused by excessive alcohol consumption are widespread throughout the human body. One of the primary consequences of alcohol abuse, particularly acute alcohol abuse, is very high levels of NADH formed from excessive ethanol oxidation. A high NADH:NAD+ ratio shifts the redox potential of the cells, shifting the normal physiological equilibrium, particularly within NAD-dependent dehydrogenase-catalyzed reactions. These particular reactions occur within various metabolic pathways, such as: citric acid cycle, glycolysis and branched-chain amino acid catabolism. As such, a disruptive effect within these metabolic pathways results in the slight accumulation of perturbation markers that can be associated with alcohol abuse. Isolation and identification of these widespread perturbation markers is difficult as they only occur in quantities only slightly higher than normal physiological values. Metabolomics makes for a very aptly used technique as it takes a holistic approach, taking into consideration the entire metabolic profile; and, with the aid of bioinformatics, is able to isolate and identify particular variables/metabolites of interest and accredit them as the variables responsible for the greatest variation between control and experimental groups. A novel approach used within this investigation effectively reduced the voluminous metabolomics data generated allowing for more efficient multivariate analysis. Application of three separate statistical models, namely: (1) Unfolding PCA, (2) Cross-sectional PCA, and (3) ANOVA Simultaneous Component Analysis (ASCA), were used for analyzing the complex 3-dimensional data set created within this acute alcohol abuse investigation. Each model presented certain strengths and difficulties. Taking into consideration the results from all 3 models, the first phase of this investigation confidently illustrates the differentiation between control cases and individuals administered an acute alcohol dose and, subsequently allow for variables responsible for this separation to be: identified as variables of importance, selected and categorized into specific pathways and, finally, labelled as perturbation markers. Through experimental observation it was noted that a large number of perturbation markers associated with the branched-chain amino acid pathway were present within the experimental cases. A hypothesis was created from this observation, re-enforcing the principle that metabolomics is a hypothesis-generating system. The subsequent second phase of this investigation involves a targeted experimental protocol aimed at evaluating the proposed hypothesis, with a focus on three secondary metabolites of the isoleucine catabolism pathway (ethylhydracrylic acid, tiglylglycine and 2-methyl-3hydroxybutyric acid). Results of this targeted approach show a definite perturbance, similar to a very minor inherited metabolic disorder, occurs within the isoleucine catabolism pathway in response to an acute alcohol dose. As to our knowledge, no information pertaining to the influences of acute alcohol abuse (or even chronic alcohol abuse) within the branched-chain amino acid pathway exists within the current literature, as of date. As such, the experimental observations presented and evaluated within this investigation provide a novel and more in-depth insight into the ethanol-induced perturbances within human metabolism.
Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.

In 2001, the WHO declared tuberculosis (TB) a global emergency, as one third of the world‟s population suffered from latent M. tuberculosis infection. Today, a decade later, millions of people still die worldwide as a result of this disease. This growing TB incidence may be ascribed to a variety of reasons, including, amongst others, the inadequacies associated with the currently available diagnostic methods and TB treatment regimes, especially when considering the growing MDR-TB and HIV epidemics. This study investigated the potential of metabolomics as a tool for characterising TB and various TB-causing bacteria, allowing for a better understanding of TB disease mechanisms, which may ultimately lead to improved diagnostic and treatment regimens. Firstly, we investigated the potential of a fatty acid, metabolomics approach to characterise various cultured Mycobacterium species. For this exploration, three fatty acid extraction procedures, prior to GC-MS analyses, were compared based on their respective repeatability and extraction capacities. Using the data obtained from the analyses done with the most optimal extraction approach (the modified Bligh-Dyer method), multivariate statistical analyses were able to differentiate between the various Mycobacterium species at a detection limit of 1 x 103 bacterial mL-1, in 16 hours. Subsequently, the compounds best describing the variation between the sample groups were identified as potential metabolite markers and were discussed in the light of previous studies. The most optimal GC-MS, fatty acid metabolomics approach, mentioned above, was then applied to analyse and characterise a wild-type M. tuberculosis parent strain and two rifampicinresistant conferring rpoB mutants (S522L and S531L). Due to the variation in their fatty acid profiles, a clear differentiation was achieved between these M. tuberculosis sample groups, and those metabolites contributing most to this variation were identified as metabolite markers characteristic for rifampicin-resistance. The altered metabolite markers detected in the rpoB mutants propose a decreased synthesis of various 10-methyl branched-chain fatty acids and cell wall lipids, and an increased use of the shorter-chain fatty acids and alkanes as alternative carbon sources. Furthermore, the rpoB S531L mutant, previously reported to occur in well over 50% of all clinical rifampicin-resistant M. tuberculosis strains, showed a better capacity for using these alternative energy sources, in comparison to the less frequently detected rpoB S522L mutant. The developed fatty acid GC-MS metabolomics approach was then successfully adapted in order to improve its speed, cost and complexity. This improved fatty acid extraction method was furthermore compared to another, similar approach (total metabolome extraction method), developed for the extraction of a much wider variety of compounds, prior to GC-MS and statistical data analyses. Although both these methods show promise for bacterial characterisation using matabolomics, the total metabolome extraction method proved the better of the two methods because it is comparatively simpler, faster (taking less than 4 hours), more repeatable, better differentiates between sample groups due to less within group variation, has a lower detection limit, and isolates a wider variety of biologically relevant metabolites (as opposed to fatty acids alone). We, furthermore, identified and described the occurrence of those compounds, extracted by both methods, which contribute most to the variation between the bacterial groups, in order to validate these methods for metabolomic applications and the isolation of compounds with biological relevance. In order to evaluate the potential of this developed metabolomics approach for application to biological samples other than bacteriological cultures, it was adapted for the direct analyses of complex sputum samples. For this application, four sputum pre-extraction preparation methods, including three standard Mycobacterium cell isolation procedures (Sputolysin, NALC-NaOH, and NaOH) and a fourth, applying only a simple ethanol homogenisation step, prior to direct sputum extraction, were compared. Of these methods, the ethanol homogenisation method proved to have the best comparative extraction efficiency, repeatability and differentiation capacity, when used in combination with the previously developed metabolomics methods. Subsequently, when applying this approach to patient collected sputum samples, a set of metabolite markers, differentiating the TB-positive from the TB-negative samples, were identified. These markers could directly be linked to: 1) the physical presence of the M. tuberculosis in these samples; 2) changes in the bacterial metabolome due to in vivo growth conditions and; 3) changes in the human metabolome due to pulmonary M. tuberculosis infection. In addition to the proposal of a number of new hypotheses, explaining various mechanisms of TB and drug-resistant TB, the mapping of the newly identified metabolite markers to known metabolic pathways led to the confirmation of various previously suggested metabolic pathways and alterations thereof due to an assortment of perturbations. Therefore, this study significantly contributes to the characterisation of various TB causing bacteria, rifampicin-resistant M. tuberculosis strains and the TB disease state, which may in future lead to the development of innovative TB vaccination, diagnostic and treatment protocols.
Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012

Biological processes in living organisms consist of a vast number of different molecular networks and interactions, which are complex and often hidden from our understanding. This work is focused on recovery of such details for two quite distant examples: acclimation to extreme freezing tolerance in Siberian spruce (Picea obovata) and detection of proteins associated with prostate cancer. The first biological system in the study, upon P. obovata, is interesting by this species ability to adapt and sustain extremely low temperatures, such as -60⁰C or below. Despite decades of investigations, the essential features and mechanisms of the amazing ability of this species still remains unclear. To enhance knowledge about extreme freezing tolerance, the metabolome and proteome of P. obovata’s needles were collected during the tree’s acclimation period, ranging from mid August to January, and have been analyzed. The second system within this study is the plasma proteome analysis of high risk prostate cancer (PCa) patients, with and without bone metastases. PCa is one of the most common cancers among Swedish men, which can abruptly develop into an aggressive, lethal disease. The diagnostic tools, including PSA-tests, are insufficient in predicting the disease’s aggressiveness and novel prognostic markers are urgently required. Both biological systems have been analyzed following similar steps: by two-dimensional difference gel electrophoresis (2D-DIGE) techniques, followed by protein identification using mass spectrometry (MS) analysis and multivariate methods. Data processing has been utilized for searching for proteins that serve as unique indicators for characterizing the status of the systems. In addition, the gas chromatography-mass spectrometry (GC-MS) study of the metabolic content of P.obovata’s needles, from the extended observation period, has been performed. The studies of both systems, combined with thorough statistical analysis of experimental outcomes, have resulted in novel insights and features for both P. obovata and prostate cancer. In particular, it has been shown that dehydrins, Hsp70s, AAA+ ATPases, lipocalin and several proteins involved in cellular metabolism etc., can be uniquely associated with acclimation to extreme freezing in conifers. Metabolomic analysis of P. obovata needles has revealed systematic metabolic changes in carbohydrate and lipid metabolism. Substantial increase of raffinose, accumulation of desaturated fatty acids, sugar acids, sugar alcohols, amino acids and polyamines that may act as compatible solutes or cryoprotectants have all been observed during the acclimation process. Relevant proteins for prostate cancer progression and aggressiveness have been identified in the plasma proteome study, for patients with and without bone metastasis. Proteins associated with lipid transport, coagulation, inflammation and immune response have been found among them.

Introduction Oesophageal cancer has a poor prognosis. Early diagnosis and the use of chemotherapy and surgery for local disease are key to improving survival. This study was designed to see if plasma and tissue metabolic profiles could be used to identify oesophago-gastric malignancy, indicate the presence of unstable pre-malignant (Barrett’s) epithelium or predict response to chemotherapy. Methods Patients were recruited from University Hospitals Birmingham between May 2009 and March 2010. Nuclear Magnetic Resonance (NMR) metabolomics was performed on filtered plasma and extracted tissue samples. Results Some 258 participants were recruited. NMR metabolomics discriminated between normal, Barrett’s and neoplastic epithelium. Tissue levels of hypoxanthine were highest in oesophageal adenocarcinoma compared to adjacent normal mucosa. Levels in Barrett’s mucosa in the presence of cancer fell between normal and neoplastic mucosa. 3-hydroxybutyrate levels were elevated both in cancer tissues and plasma compared to controls. Plasma levels of 3-hydroxybutyrate were higher in patients with node positive and full thickness tumours compared to those who were node negative with early local disease. Conclusion NMR metabolomics identified metabolic profiles that characterized different histologic tissue types. Metabolites involved in oesophageal carcinogenesis might influence diagnostic and management strategies in these patients.

Age-related macular degeneration (AMD) is a major cause of irreversible central sight loss in the elderly. Many factors affect disease onset and progression, and these include age, environmental stressors such as smoking, diet, inflammation and genetic polymorphisms, all of which are likely to influence metabolism. In some complex diseases, metabolomics, which involves the identification of a metabolic fingerprint in a biofluid or tissue, has been shown to discriminate metabolic changes associated with different disease processes and to identify specific phenotypes. We have applied metabolomics, in the first study of its kind, to analyse, using NMR spectroscopy, both serum and urine metabolic characteristics in patients with dry and wet AMD (n=104). NMR spectral analysis showed good clustering as well as separation among the serum and urine from dry and wet AMD patients. The results show that metabolite profiles can distinguish dry and wet AMD, but that the pathways involved, glycolysis, urea cycle and Kreb’s cycle, are involved in both forms of the disease. It is likely that the pathogenesis of dry and wet AMD is similar and that the severity of ocular damage and systemic inflammation would account for the distinguishing profiles. These data support the use of metabolomics in identifying biological pathways involved in pathogenesis of AMD, but not in the diagnosis or prognosis of disease.

Despite the clinical importance of hypertrophic cardiomyopathy (HCM) and a recognition that aberrant energetics contributes to its pathogenesis, significant uncertainty remains regarding the determinants of the clinical phenotype. The central hypothesis underlying this thesis is that the clinical manifestations of HCM are causatively influenced by perturbations in cellular metabolism, and that the metabolome in HCM, both at rest and during stress, is distinct from that of healthy hearts and other cardiac diseases manifesting left ventricular hypertrophy (LVH). Aortic stenosis, the most common valvular heart disease in the western world, and often presenting with a prominent phenotype of LVH, was the principal comparator disease state. The metabolome of coronary sinus effluent from densely phenotyped patients with HCM, aortic stenosis with LVH and controls was systematically studied. These patients were assessed under different physiological states (at rest and during stress induced by pacing). The present work focused on fatty acid, carbohydrate and amino acid metabolism. The metabolomic changes identified were then validated by metabolomic and an orthogonal transcriptomic analysis of genes regulating these metabolic pathways in separately recruited patient cohorts. Most prominently, this study identified substantial metabolic dysregulation in aortic stenosis, consisting of impaired fatty acid β-oxidation at rest and a trend towards reduced uptake of citric acid cycle intermediates and branched chain amino acids (BCAAs) during chronotropic stress (pacing). Transcriptomic analysis revealed only partial and ineffective upregulation in PPARA expression, with a corresponding downregulation of several PPARα target genes regulating fatty acid metabolism. In contrast, there was a seemingly appropriate upregulation of citric acid cycle activity and BCAA metabolism in HCM patients during stress. During discovery and validation stages selected metabolites differentiated the control vs aortic stenosis and HCM vs aortic stenosis cohorts with relatively similar and high area under the curve (AUC) values on receiver operating characteristic (ROC) analysis. This work presented in this thesis represents the first comprehensive analysis of changes in metabolic pathways (transcriptional to metabolome) in aortic stenosis, and its comparison to HCM. The findings represent a significant increase in our understanding of the underlying metabolic derangements accompanying these disorders and provide us with promising avenues into potential therapeutic targets (e.g. PPARα), with strong clinical translation potential.

Doutoramento em Química
Chapter 1 introduces the scope of the work by identifying the clinically relevant prenatal disorders and presently available diagnostic methods. The methodology followed in this work is presented, along with a brief account of the principles of the analytical and statistical tools employed. A thorough description of the state of the art of metabolomics in prenatal research concludes the chapter, highlighting the merit of this novel strategy to identify robust disease biomarkers. The scarce use of maternal and newborn urine in previous reports enlightens the relevance of this work. Chapter 2 presents a description of all the experimental details involved in the work performed, comprising sampling, sample collection and preparation issues, data acquisition protocols and data analysis procedures. The proton Nuclear Magnetic Resonance (NMR) characterization of maternal urine composition in healthy pregnancies is presented in Chapter 3. The urinary metabolic profile characteristic of each pregnancy trimester was defined and a 21-metabolite signature found descriptive of the metabolic adaptations occurring throughout pregnancy. 8 metabolites were found, for the first time to our knowledge, to vary in connection to pregnancy, while known metabolic effects were confirmed. This chapter includes a study of the effects of non-fasting (used in this work) as a possible confounder. Chapter 4 describes the metabolomic study of 2nd trimester maternal urine for the diagnosis of fetal disorders and prediction of later-developing complications. This was achieved by applying a novel variable selection method developed in the context of this work. It was found that fetal malformations (FM) (and, specifically those of the central nervous system, CNS) and chromosomal disorders (CD) (and, specifically, trisomy 21, T21) are accompanied by changes in energy, amino acids, lipids and nucleotides metabolic pathways, with CD causing a further deregulation in sugars metabolism, urea cycle and/or creatinine biosynthesis. Multivariate analysis models´ validation revealed classification rates (CR) of 84% for FM (87%, CNS) and 85% for CD (94%, T21). For later-diagnosed preterm delivery (PTD), preeclampsia (PE) and intrauterine growth restriction (IUGR), it is found that urinary NMR profiles have early predictive value, with CRs ranging from 84% for PTD (11-20 gestational weeks, g.w., prior to diagnosis), 94% for PE (18-24 g.w. pre-diagnosis) and 94% for IUGR (2-22 g.w. pre-diagnosis). This chapter includes results obtained for an ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) study of pre-PTD samples and correlation with NMR data. One possible marker was detected, although its identification was not possible. Chapter 5 relates to the NMR metabolomic study of gestational diabetes mellitus (GDM), establishing a potentially predictive urinary metabolic profile for GDM, 2-21 g.w. prior to diagnosis (CR 83%). Furthermore, the NMR spectrum was shown to carry information on individual phenotypes, able to predict future insulin treatment requirement (CR 94%). Chapter 6 describes results that demonstrate the impact of delivery mode (CR 88%) and gender (CR 76%) on newborn urinary profile. It was also found that newborn prematurity, respiratory depression, large for gestational age growth and malformations induce relevant metabolic perturbations (CR 82-92%), as well as maternal conditions, namely GDM (CR 82%) and maternal psychiatric disorders (CR 91%). Finally, the main conclusions of this thesis are presented in Chapter 7, highlighting the value of maternal or newborn urine metabolomics for pregnancy monitoring and disease prediction, towards the development of new early and non-invasive diagnostic methods.
O Capítulo 1 descreve o enquadramento deste trabalho identificando as doenças pré-natais relevantes e os métodos de diagnóstico actualmente disponíveis. É depois apresentada a metodologia seguida, assim como uma breve introdução dos princípios dos métodos analíticos e estatísticos aplicados. O capítulo é concluído com uma descrição do estado da arte na área de metabolómica em investigação pré-natal, identificando o mérito desta inovadora estratégia para a identificação de marcadores robustos de doenças pré-natais. A relevância deste trabalho torna-se clara através do escasso uso de urina materna e do recém-nascido em trabalhos anteriores. O Capítulo 2 descreve os procedimentos experimentais utilizados neste trabalho, incluindo condições de amostragem, recolha e preparação das amostras, protocolos de aquisição e de tratamento dos dados. A caracterização da composição da urina materna, através de espectroscopia de Ressonância Magnética Nuclear (RMN) de protão é apresentada no Capítulo 3. Define-se o perfil metabólico urinário característico para cada trimestre de gravidez, tendo sido encontrado um conjunto de 21 metabolitos descritivo das alterações metabólicas ocorridas ao longo da gravidez. 8 metabolitos foram encontrados a variar com a gravidez, pela primeira vez, tendo sido confirmadas variações metabólicas conhecidas. É ainda estudado o efeito do não-jejum (usado neste trabalho) como possível factor de confusão. O Capítulo 4 apresenta o estudo metabolómico de urina materna do 2º trimestre para o diagnóstico de doenças fetais e previsão de complicações mais tarde desenvolvidas. Este estudo compreende a aplicação de um método de selecção de variáveis desenvolvido no âmbito desta tese. Observou-se que as malformações fetais (e, especificamente, do sistema nervoso central, SNC) e as cromossomopatias (e, especificamente, a trissomia 21, T21) são acompanhadas por alterações nos metabolismos energético, dos aminoácidos, lípidos e nucleótidos, enquanto que as cromossomopatias mostraram ser acompanhadas por uma desregulação adicional dos metabolismos dos açúcares, ciclo da ureia e/ou biossíntese da creatinina. A validação dos modelos multivariados revelou taxas de classificação (CR) de 84% para malformações (87%, SNC) e 85% para CD (94%, T21). Para o parto pré-termo, pré-eclampsia (PE) e restrição de crescimento intrauterino (RCIU) observaram-se perfis que podem ajudar à previsão precoce, com CR 84% para pretermo (11-20 semanas de gestação, g.w. pré-diagnóstico), 94% para PE (18-24 g.w. pré-diagnóstico) e 94% para RCIU (2-22 g.w. pré-diagnóstico). Este capítulo inclui resultados obtidos por cromatografia líquida de ultra eficiência acoplada a espectrometria de massa (UPLC-MS) para pré-pretermo e correlação com os dados de RMN. Um possível composto marcador foi detectado mas a sua identificação não foi possível. O Capítulo 5 descreve o estudo metabolómico por RMN da diabetes mellitus gestacional (DMG), estabelecendo-se um perfil metabólico potencialmente preditivo da doença (CR 83%, 2-21 g.w. pré-diagnóstico). Verificou-se ainda que o espectro de RMN contém informação sobre o fenótipo individual, capaz de prever a necessidade futura de tratamento com insulina (CR 94%). No Capítulo 6 demonstra-se o impacto do tipo de parto (CR 88%) e género do bebé (CR 76%) no perfil da urina do recém-nascido. Verificou-se ainda que a prematuridade, depressão respiratória, crescimento grande para a idade gestacional e malformações induzem perturbações metabólicas relevantes (CR 82-92%), assim como algumas doenças maternas como a DMG (CR 82%) e doenças psiquiátricas (91% CR). Finalmente, no Capítulo 7 apresentam-se as principais conclusões deste trabalho, enfatizando o potencial da metabolómica de urina materna e do bebé para o acompanhamento da gravidez e previsão de doenças, visando o desenvolvimento de novos métodos de diagnóstico precoce e não-invasivo.

Mestrado em Biotecnologia - Biotecnologia Molecular
Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by massive loss of dopaminergic neurons. Despite of decades of research the cause of sporadic PD is still unknown. PD is a complex multifactorial disorder, which probably results from an elaborate interplay of mostly unknown factors: several genes, modifying effects by susceptibility alleles, environmental exposures and gene-environment interactions, and their direct impact on the developing and aging brain. The discovery of disease-related genes has contributed substantially to the understanding of the molecular mechanisms involved in PD pathogenesis. It is known that a cascade of events leading to cell death, including the oxidative stress, contributes for the pathogenesis of PD. Among several genes mutated in familial PD, only DJ-1, an autosomal recessive gene causative of familial early onset PD, plays a direct role in oxidative defense mechanisms of substantia nigra pars compacta. The study of DJ-1 biology can provide important clues to altered cellular pathways in PD. Thus, understanding how the causative DJ-1 mutations interfere with the structure and function of DJ-1 protein is of critical importance. Mutations in DJ-1 gene may lead to loss of neuroprotective function of the protein. In this way it may occur a homeostatic imbalance in cell system and metabolites, which can be used as cellular markers of stress conditions. Therefore, the aim of this study was to compare multiple biological conditions to identify the metabolites that are significantly altered in resting and oxidative stress conditions, and access also the effect of the addition of the recombinant DJ-1 WT and mutants to SH-SY5Y cell line under normal and oxidative stress conditions. In order to achieve this goal, different recombinant protein mutants were produced and structurally characterized to access their rule in metabolite modulation. Once added to cells, an untargeted mass spectrometry analysis of metabolites was conducted in order to find potential and putative metabolites of interest. This was the first study for oxidative stress metabolomics profiling with the exogenous addition of recombinant DJ-1 WT and mutants and allowed the finding of eight possible oxidative stress biomarkers. In the future, these results must be validated in a targeted analysis, for metabolite ID verification, quantitation, functional interpretation, and pathway analysis, to try to understand their modulation by DJ-1 and their potential use as oxidative stress markers and latter as Parkinson´s disease biomarkers. Hence, these findings may contribute to future strategies for the treatment and prevention of the disease and offer new directions for recognizing disease-specific biochemical indicators.
A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum caracterizada pela perda massiva de neurónios dopaminérgicos. Apesar de décadas de pesquisa a causa da DP esporádica ainda é desconhecida. A DP é uma doença multifactorial complexa, que provavelmente resulta de uma interacção elaborada na sua maioria de factores desconhecidos: vários genes, efeitos modificadores de alelos de susceptibilidade, exposições ambientais e interacções gene-ambiente e o seu impacto directo sobre o desenvolvimento e envelhecimento do cérebro. A descoberta de genes relacionados com a doença tem contribuído substancialmente para a compreensão dos mecanismos moleculares envolvidos na patogénese da DP. Sabe-se que um conjunto de acontecimentos que conduzem à morte da célula, incluindo o stress oxidativo, contribui para a patogénese da DP. Entre os vários genes mutados na DP familiar, apenas o gene DJ-1, um gene autossómico recessivo causador de DP familiar de início precoce, desempenha um papel directo nos mecanismos de defesa oxidativa da substantia nigra pars compacta. O estudo da biologia da DJ-1 pode fornecer informações importantes para vias celulares alteradas na DP. Assim, a compreensão de como as mutações da DJ-1 interferem com a estrutura e função da proteína é de crucial importância. Mutações no gene DJ -1 podem levar à perda da função neuroprotectora da proteína. Deste modo, pode ocorrer um desequilíbrio homeostático no sistema e nos metabolitos celulares, que podem ser utilizados como marcadores celulares de condições de stress. Portanto, o objectivo deste estudo foi comparar várias condições biológicas para identificar metabolitos que são significativamente alterados em condições de repouso e de stress oxidativo, e avaliar também o efeito da adição da DJ-1 WT e mutantes recombinantes à linha celular SH-SY5Y sob condições normais e condições de stress oxidativo. A fim de atingir esse objectivo, diferentes proteínas mutantes recombinantes foram produzidas e caracterizadas estruturalmente para avaliar a sua acção na modulação dos metabolitos. Uma vez adicionadas às células, uma análise não direccionada de espectrometria de massa dos metabolitos foi realizada a fim de encontrar potenciais metabolitos de interesse. Este foi o primeiro estudo para o perfil metabolómico do stress oxidativo com a adição exógena de DJ-1 WT e mutantes recombinantes, e permitiu a descoberta de oito possíveis biomarcadores de stress oxidativo. No futuro, estes resultados devem ser validados numa análise direccionada, para identificação, quantificação, interpretação funcional e análise das vias dos metabolitos, para tentar compreender a sua modulação pela DJ-1 e o seu potencial uso como marcadores de stress oxidativo, e em último caso como biomarcadores da doença de Parkinson. Assim, estes resultados podem contribuir para estratégias futuras para o tratamento e prevenção da doença e oferecer novos rumos para o reconhecimento de indicadores bioquímicos específicos da doença.

This thesis focused on defining the urinary reference values and investigating the urinary proteome and metabolome of captive giraffes (Giraffa camelopardalis) by using a non-invasive sampling method. Urine samples were collected with a syringe from the ground, immediately after spontaneous voiding, by aspirating the upper part of samples, to avoid them to come into contact with the soil contaminants as much as possible. To prove the sampling’s reliability, cow urines were used and the results of both types of samples (those collected in sterile urine cups and those collected from the ground with a syringe) were compared. This experiment revealed no statistical differences in the variables investigated (urine total protein, uTP; urine creatinine, uCrea; urine protein:creatinine ratio, UPC; number of protein bands and band protein quantification detected by 1D-electrophoresis-SDS-PAGE), which proved the reliability of this sampling technique. By establishing the urinary reference values as well as by identifying the urinary proteome, it was possible to obtain information about the renal function in giraffes for the first time. Just like other ruminants, urine of giraffes contains low quantity of proteins, the majority of which shows low molecular mass (MM). The proteins identified in their urines might act as a defence against microbes and play a role in the ability of giraffes to concentrate urine. A first insight into the urinary metabolome allowed to identify and quantify 39 molecules; this provided some information on some physiological adaptations of giraffes and were influenced by sex and age.

The researcher studied the role of amino acid metabolism in osteoarthritis progression. The study suggests that this abnormal amino acid metabolism aids in the development of the disease. This data further suggests that amino acids could be potential circulatory markers for diagnosing OA and therapeutic strategies of amino acids supplementation could be considered as a potential treatment.

El càncer d’endometri és la neoplàsia ginecològica més freqüent en els països desenvolupats. Malgrat que la majoria dels carcinomes són curables amb un tractament adequat, al voltant del 20% dels tumors es comporten de forma agressiva i suposen un repte terapèutic. És per aquest motiu que sorgeix la necessitat d’identificar nous paràmetres per tal de seleccionar pacients amb risc de recidiva o metàstasi. La cèl•lula eucariota presenta canvis en el seu metabolisme com a resposta coordinada a diferents situacions fisiològiques i patològiques, entre elles el càncer. L’anàlisi del metaboloma, mitjançant la metabolòmica, pot permetre identificar metabòlits diferencials que representen el producte final de les vies de senyalització que estan alterades en el càncer. Per aquest motiu hem volgut realitzar una anàlisi metabolòmica del càncer d’endometri. A més a més, per tal de validar els resultats i traslladar-los a la pràctica clínica s’han avaluat els nivells d’expressió dels metabòlits diferencials més significatius en arrays de teixit (TMAs). En primer lloc, els resultats han mostrat que el procés de carcinogènesi del càncer d’endometri defineix un perfil metabolòmic específic. Els resultats suggereixen que la via dels endocannabinoides pot estar implicada en la gènesi i progressió del carcinoma endometrioide. Així mateix, l’alteració del metabolisme de les purines pot estar implicada en fenòmens d’invasió miometrial en el càncer d’endometri. En segon lloc, l’estudi metabolòmic ha mostrat un perfil diferencial entre carcinomes endometrioides i serosos. A més a més, ha permès identificar dues molècules, ADI1 i BCAT1, que poden estar implicades en la gènesi de les neoplàsies endometrioides i en la progressió tumoral. Així mateix, aquests compostos poden ajudar a establir el diagnòstic diferencial entre aquests dos subtipus histològics amb pronòstics molt diferents. Finalment, partint de la premissa que l’angiogènesi és un mecanisme essencial per al creixement, invasió i disseminació tumoral i que els carcinomes d’endometri amb flux sanguini intratumoral disminuït tenen pitjor pronòstic, s’ha analitzat el perfil metabolòmic del càncer d’endometri en funció del seu flux sanguini. El resultats mostren un perfil metabolòmic específic dels tumors segons el seu flux sanguini i permeten identificar Resolvina D i fosfolípids específics diferencials entre tumors d’alt i baix flux sanguini. Aquestes molècules poden estar implicades en l’angiogènesi i progressió tumoral en el càncer d’endometri.
El cáncer de endometrio es la neoplasia ginecológica más frecuente en los países desarrollados. A pesar de que la mayoría de los carcinomas son curables con un tratamiento adecuado, alrededor del 20% de los tumores se comportan de forma agresiva y suponen un reto terapéutico. Por este motivo surge la necesidad de identificar nuevos parámetros que permitan seleccionar pacientes con riesgo de recidiva o metástasis. La célula eucariota presenta cambios en su metabolismo como respuesta coordinada a diferentes situaciones fisiológicas y patológicas, entre ellas el cáncer. El análisis del metaboloma, mediante la metabolómica, puede ayudar a identificar metabolitos diferenciales que representan el producto final de las vías de señalización que están alteradas en el cáncer. Por esta razón hemos querido realizar un análisis metabolómico del cáncer de endometrio. Además, para la validación de los resultados y su translación a la práctica clínica se han evaluado los niveles de expresión de los metabolitos diferenciales más significativos en arrays de tejido (TMAs). En primer lugar, los resultados han mostrado que el proceso de carcinogénesis del cáncer de endometrio define un perfil metabolómico específico. Los resultados sugieren que la vía de los endocannabinoides puede estar implicada en la génesis y progresión del carcinoma endometrioide. Además, la alteración del metabolismo de las purinas puede estar implicada en fenómenos de invasión miometrial en el cáncer de endometrio. En segundo lugar, el estudio metabolómico ha mostrado un perfil diferencial entre carcinomas endometrioides y serosos. Además ha permitido identificar dos moléculas, ADI1 i BCAT1, que pueden estar implicadas en la génesis de las neoplasias endometrioides así como en la progresión tumoral. Asimismo, estos dos compuestos pueden ser útiles en el diagnóstico diferencial de estos dos subtipos histológicos con pronósticos tan distintos. Finalmente, partiendo de la base que la angiogénesis es un mecanismo esencial para el crecimiento, invasión y diseminación tumoral y que los carcinomas de endometrio con flujo sanguíneo intratumoral disminuido tienen peor pronóstico, se ha analizado el perfil metabolómico del cáncer de endometrio en función de su flujo sanguíneo. Los resultados muestran un perfil metabolómico específico de los tumores según su flujo sanguíneo y permiten identificar Resolvina D i fosfolípidos específicos diferenciales entre tumores de alto y bajo flujo sanguíneo. Estas moléculas pueden estar implicadas en la angiogénesis y progresión tumoral en el cáncer de endometrio.
Endometrial cancer is the most frequent gynecological malignancy in developed countries. Although most carcinomas are curable with adequate treatment, about 20% of tumors behave aggressively and pose a therapeutic challenge. For this reason, there is a need to identify new parameters that allow the selection of a patient with risk of recurrence or metastasis. The eukaryotic cell presents changes in its metabolism as a coordinated response to different physiological and pathological situations, including cancer. The analysis of the metabolome, through metabolomics, can help identify differential metabolites that represent the final product of the signaling pathways that are altered in cancer. For this reason we wanted to perform a metabolomic analysis of endometrial cancer. In addition, for the validation of the results and their translation to clinical practice, the expression levels of the most significant differential metabolites have been evaluated using tissue arrays (TMAs). First, the results have shown that the process of carcinogenesis of endometrial cancer defines a specific metabolomic profile. The results suggest that the endocannabinoid pathway may be involved in the genesis and progression of endometrioid carcinoma. In addition, the alteration of the purine metabolism may be involved in myometrial invasion phenomens in endometrial cancer. Second, the metabolomic study has shown a differential profile between endometrioid and serous carcinomas and has allowed the identification of two molecules, ADI1 and BCAT1, which may be involved in the genesis of endometrioid neoplasms as well as in tumor progression. Likewise, these two compounds can be useful in the differential diagnosis of these two histological subtypes with such different prognoses. Finally, starting from the basis that angiogenesis is an essential mechanism for tumor growth, invasion and dissemination and that endometrial carcinomas with decreased intratumoral blood flow have a worse prognosis, the metabolomic profile of endometrial carcinoma has been analysed according to its blood flow. The results show a specific metabolomic profile of the tumors according to their blood flow and allow identifying Resolvin D and specific phospholipids differentials between high and low blood flow tumors. These molecules may be involved in angiogenesis and tumor progression in endometrial cancer.

L’artritis inflamatòria representa una gran càrrega social i econòmica tot i els recents avenços terapèutics. Malgrat un millor coneixement dels mecanismes patogénics, l’elecció de l’tractament encara es realitza a manera de prova, el que condueix a una manca de control de l’activitat de la malaltia en aproximadament el 30% dels pacients i una alta taxa d’efectes secundaris. La identificació de mediadors de malaltia i predictors de resposta a el tractament és necesaria per permetre el tractament adequat i aconseguir la remissió clínica o al menys una baixa activitat de la malaltia. És poc prob-able que un sol biomarcador proporcioni informació suficient per explicar aquestes malalties heterogènies. La metabolòmica és una eina que es pot utilitzar per al descobriment de biomarcadors, ja que pot identificar perfils d’una gran quantitat de metabòlits en diferents tipus de mostres. Els metabòlits no són només el resultat final dels processos químics que tenen lloc en la cèl·lula, sinó que també juguen un paper crític en una varietat de processos cel·lulars, com les modificacions postranslacionals i la regulació de les cèl·lules immunes. Amb la hipòtesi que els metabòlits circulants reflecteixen processos sinovials, aquest projecte va tenir com a objectiu estudiar els metabòlits circulants en relació amb l’activitat de la enfermedad y la resposta als fàrmacs antireumàtics modificadors de la malaltia. Descrivim diferents perfils metabolòmics en pacients amb artritis inflamatòria comparats amb controls. També identifiquem metabòlits que es correlacionen amb l’activitat de la malaltia i que poden ser mediadors de la malaltia, així com metabòlits associats amb la resposta a el tractament.
La artritis inflamatoria representa una gran carga social y económica a pesar de los recientes avances terapéuticos. A pesar de un mejor conocimiento de los mecanismos patogénicos, la elección del tratamiento todavía se realiza a modo de prueba, lo que conduce a una falta de control de la actividad de la enfermedad en aproximadamente el 30 % de los pacientes y una alta tasa de efectos secundarios. La identificación de mediadores de enfermedad y predictores de respuesta al tratamiento es necesaria para permitir el tratamiento adecuado y lograr la remisión clínica o al menos una baja actividad de la enfermedad. Es poco probable que un solo biomarcador proporcione información suficiente para explicar estas enfermedades heterogéneas. La metabolómica es una herramienta que se puede utilizar para el descubrimiento de biomarcadores, ya que puede identificar perfiles de una gran cantidad de metabolitos en diferentes tipos de muestras. Los metabolitos no son solo el resultado final de los procesos químicos que ocurren en la célula, sino que también juegan un papel crítico en una variedad de procesos celulares, como las modificaciones postranslacionales y la regulación de las células inmunes. Con la hipótesis de que los metabolitos circulantes reflejan procesos sinoviales, este proyecto tuvo como objetivo estudiar los metabolitos circulantes en relación con la actividad de la enfermedad y la respuesta a los fármacos antirreumáticos modificadores de la enfermedad. Describimos diferentes perfiles metabolómicos en pacientes con artritis inflamatoria comparados con controles. También identificamos metabolitos que se correlacionan con la actividad de la enfermedad y que pueden ser mediadores de la enfermedad, asi como metabolitos asociados con la respuesta al tratamiento.
Inflammatory arthritis represents a great social and economic burden despite recent therapeutic advances. Although we have a better understanding of the pathogenic mechanisms, treatment election is still made on a trial basis, which leads to lack of control of disease activity in approximately 30% of patients and a high rate of side effects. The identification of disease mediators and predictors of response to treatment are needed to allow the adequate treatment and achieve clinical remission or at least low disease activity. A single biomarker is unlikely to provide sufficient information to explain these heterogeneous diseases. Metabolomics is a tool that can be used for biomarker discovery as it can identify profiles of a large number of metabolites in different types of samples. Metabolites are not just the end result of chemical processes that occur in the cell, but also play critical role in a variety of cellular processes, such as post-translational modifications and immune cell regulation. With the hypothesis that circulating metabolites reflect synovial processes, this project aimed to study circulating metabolites in relation to disease activity and response to disease modifying anti- rheumatic drugs. We described different metabolomic profiles in patients with inflammatory arthritis compared to controls. We also identified metabolites that correlate with disease activity and that may be mediators of disease, as well as metabolites that are associated with response to treatment.
Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina